Compositions and related methods for modulating endosymbionts

ABSTRACT

Provided herein are methods and compositions for modulating the fitness of a host invertebrate (e.g., insect, mollusk, or nematode) by altering interactions between the host and one or more micoorganisms resident in the host. The invention features a composition including a modulating agent (e.g., a polypeptide, nucleic acid, small molecule, or combinations thereof) that can induce changes in the host&#39;s microbiota in a manner that modulates (e.g., increases or decreases) host fitness. The modulating agent described herein may modulate the fitness of a variety of invertebrates that are important for agriculture, commerce, and/or public health.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/463,451, filed on Feb. 24, 2017, and U.S. Provisional Application No.62/583,990, filed on Nov. 9, 2017, the contents of which are herebyincorporated herein by reference in their entireties.

BACKGROUND

Invertebrate organisms (e.g., insects, mollusks, or nematodes) arepervasive in the human environment. In some instances, invertebratesserve beneficial roles, such as nematodes or arthropods utilized inagriculture for pollination efforts and pest control or in commerce forthe production of commercial products (e.g., honey or silk). In otherinstances, invertebrates can be detrimental, including some species ofmollusks (e.g., snails and slugs), nematodes, or insects that can beserious crop pests or carriers of disease. Thus, there is need in theart for methods and compositions to modulate the fitness ofinvertebrates that are important in agriculture, commerce, or publichealth.

SUMMARY OF THE INVENTION

Disclosed herein are compositions and methods for modulating the fitnessof invertebrates, including insects, nematodes, or mollusks, by alteringthe interactions between the host and one or more microorganismsresident in the host.

In one aspect, provided herein is a method for decreasing the fitness ofan insect, the method including delivering to the insect an effectiveamount of a polynucleotide that includes a dsRNA that decreasesexpression of an insect bacteriocyte regulatory gene or an insectimmunoregulatory gene in the insect relative to an insect that has notbeen administered the dsRNA.

In some embodiments, the gene encodes a protein from thebacteriocyte-specific cysteine rich proteins BCR family, a protein fromthe secreted proteins SP family, BicD (Protein bicaudal D), Cact(cactus), DIF (Dorsal related immunity factor), Toll (Toll InteractingProtein), or imd (immune deficiency protein). In some embodiments, thegene encodes a protein having at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid sequenceidentity to a protein listed in Table 5, Table 8, or Table 9. In someembodiments, the gene encodes a functional homolog of a protein listedin Table 5, Table 8, or Table 9. For example, the gene may encode acactus-like protein in aphids (e.g., any one of the proteins describedby GenBank Accession Nos: XP_022175228.1, XP_016656687.1,NP_001156668.1, XP_008179071.1, or XP_016656686.1, the associated aminoacid and nucleotide sequences of which are incorporated by reference).

In some embodiments, the dsRNA is complementary to 10 to 30 nucleotidesof the gene in the insect (e.g., 10 to 30 nucleotides, 12 to 28nucleotides, 14 to 26 nucleotides, 16 to 24 nucleotides, 14 to 22nucleotides, or 18 to 20 nucleotides). In some embodiments, the dsRNA iscomplementary to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides of the gene in the insect. Insome embodiments, the entire length of the dsRNA is complementary to thegene. In alternative embodiments, only a portion of the dsRNA iscomplementary to the gene.

In some embodiments, the method is effective to decrease expression ofthe gene in the insect, e.g., by about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or greater relative to aninsect that has not been administered the polynucleotide. In someembodiments, the method is effective to decrease expression of the genein the insect, e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% or greater as compared to a referencelevel (e.g., as compared to expression of one or more control genes(e.g., a housekeeping gene), expression of the same gene in a differentsample (e.g., one or more control samples), or expression of the samegene in the same sample at one or more earlier time points).

In some embodiments, the method is effective to decrease expression ofthe gene in the insect, e.g., by about 1.5×, 1.75×, 2×, 3×, 4×, 5×, 6×,7×, 8×, 9×, 10×, 25×, 50×, 75×, or 100× fold less relative to an insectthat has not been administered the polynucleotide. In some embodiments,the method is effective to decrease expression of the gene in theinsect, e.g., by about 1.5×, 1.75×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×,25×, 50×, 75×, or 100× fold less as compared to a reference level (e.g.,as compared to expression of one or more control genes (e.g., ahousekeeping gene), expression of the same gene in a different sample(e.g., one or more control samples), or expression of the same gene inthe same sample at one or more earlier time points).

In some embodiments, the method is effective to inhibit expression ofthe gene in the insect or to decrease expression of the gene to anundetectable level.

In some embodiments, the method is effective to decrease the level,diversity, or metabolism of one or more microorganisms resident in theinsect relative to an insect that has not been delivered thepolynucleotide. In some embodiments, the method is effective to decreasethe level, diversity, or metabolism of one or more microorganismsresident in the insect, e.g., by about 1.5×, 1.75×, 2×, 3×, 4×, 5×, 6×,7×, 8×, 9×, 10×, 25×, 50×, 75×, or 100× fold less relative to an insectthat has not been delivered the polynucleotide. In some embodiments, themethod is effective to decrease the level, diversity, or metabolism ofone or more microorganisms resident in the insect, e.g., by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%less relative to an insect that has not been delivered thepolynucleotide. In certain embodiments, the one or more microorganismsis a Buchnera spp.

In some embodiments, the method is effective to decrease the fitness ofthe insect relative to an insect that has not been delivered thepolynucleotide. In some embodiments, the method is effective to decreasethe fitness of the insect, e.g., by about 1.5×, 1.75×, 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, 25×, 50×, 75×, or 100× fold less relative to aninsect that has not been delivered the polynucleotide. In someembodiments, the method is effective to decrease the fitness of theinsect, e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% less relative to an insect that has not beendelivered the polynucleotide.

In some embodiments, the polynucleotide is delivered in a compositionformulated for delivery to insects. In some embodiments, the deliveryincludes delivering the polynucleotide to at least one habitat where theinsect pest grows, lives, reproduces, feeds, or infests. In someembodiments, the delivery comprises spraying the antimicrobial peptideon an agricultural crop. In some embodiments, the polynucleotide isdelivered as an insect comestible composition for ingestion by theinsect.

In some embodiments, the polynucleotide is formulated with anagriculturally acceptable carrier as a liquid, a solid, an aerosol, apaste, a gel, or a gas composition.

In some embodiments, the insect is an aphid.

In another aspect, provided herein is a composition including apolynucleotide that includes a dsRNA formulated for delivery to aninsect, wherein the dsRNA is complementary to 15 to 30 nucleotides of aninsect bacteriocyte regulatory gene or an insect immunoregulatory gene.In some embodiments, the gene encodes a protein selected from the groupconsisting of a protein from the bacteriocyte-specific cysteine richproteins BCR family, a protein from the secreted proteins SP family,BicD (Protein bicaudal D), Cact (cactus), DIF (Dorsal related immunityfactor), Toll (Toll Interacting Protein), and imd (immune deficiencyprotein). In some embodiments, the gene encodes a protein having atleast 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% amino acid sequence identity to a protein listed in Table5, Table 8, or Table 9. In some embodiments, the gene encodes afunctional homolog of a protein listed in Table 5, Table 8, or Table 9.For example, the gene may encode a cactus-like protein in aphids (e.g.,any one of the proteins described by GenBank Accession Nos:XP_022175228.1, XP_016656687.1, NP_001156668.1, XP_008179071.1, orXP_016656686.1, the associated amino acid and nucleotide sequences ofwhich are incorporated by reference). In some embodiments, the dsRNA iscomplementary to 10 to 30 nucleotides of the gene in the insect (e.g.,10 to 30 nucleotides, 12 to 28 nucleotides, 14 to 26 nucleotides, 16 to24 nucleotides, 14 to 22 nucleotides, or 18 to 20 nucleotides). In someembodiments, the dsRNA is complementary to 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides ofthe gene in the insect. In some embodiments, the entire length of thedsRNA is complementary to the gene. In alternative embodiments, only aportion of the dsRNA is complementary to the gene.

In a further aspect, provided herein is a plant comprising a topicalapplication of the compositions described herein.

In yet another aspect, provided herein is a transgenic plant cell havingin its genome a recombinant DNA construct, wherein the recombinant DNAconstruct includes a heterologous promoter operably linked to a DNAencoding a RNA including at least one double-stranded RNA region, atleast one strand of which includes a nucleotide sequence that iscomplementary to 15 to 30 nucleotides of an insect bacteriocyteregulatory gene or an insect immunoregulatory gene. In some embodiments,the gene encodes a protein selected from the group consisting of aprotein from the bacteriocyte-specific cysteine rich proteins BCRfamily, a protein from the secreted proteins SP family, BicD (Proteinbicaudal D), Cact (cactus), DIF (Dorsal related immunity factor), Toll(Toll Interacting Protein), and imd (immune deficiency protein). In someembodiments, the gene encodes a protein having at least 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% aminoacid sequence identity to a protein listed in Table 5, Table 8, or Table9. In some embodiments, the gene encodes a functional homolog of aprotein listed in Table 5, Table 8, or Table 9. For example, the genemay encode a cactus-like protein in aphids (e.g., any one of theproteins described by GenBank Accession Nos: XP_022175228.1,XP_016656687.1, NP_001156668.1, XP_008179071.1, or XP_016656686.1, theassociated amino acid and nucleotide sequences of which are incorporatedby reference). In some embodiments, the dsRNA is complementary to 10 to30 nucleotides of the gene in the insect (e.g., 10 to 30 nucleotides, 12to 28 nucleotides, 14 to 26 nucleotides, 16 to 24 nucleotides, 14 to 22nucleotides, or 18 to 20 nucleotides). In some embodiments, the dsRNA iscomplementary to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides of the gene in the insect. Insome embodiments, the entire length of the dsRNA is complementary to thegene. In alternative embodiments, only a portion of the dsRNA iscomplementary to the gene.

In yet another aspect, provided herein are compositions that include amodulating agent (e.g., a polypeptide (e.g., antibody, bacteriocin,antimicrobial peptide, or bacteriocyte regulatory peptide), a nucleicacid (e.g., DNA, RNA (e.g., mRNA, gRNA, or inhibitory RNA (e.g., RNAi,shRNA, miRNA)), CRISPR nucleic acid), a small molecule (e.g.,prostaglandin), or a combination thereof) that modulates (e.g.,increases or decreases) the fitness of an invertebrate host (e.g.,insect, mollusk, or nematode), wherein the modulating agent altersinteractions between the host and one or more microorganisms resident inthe host.

In some embodiments, the modulating agent (e.g., a polypeptide (e.g.,antibody, bacteriocin, antimicrobial peptide, or bacteriocyte regulatorypeptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA, orinhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or a combination thereof) targetsone or more host pathways that mediate interactions between the host andthe one or more microorganisms resident in the host (e.g.,host-microbiota interactions). In certain embodiments, the targeting(e.g., upregulation, downregulation, or inhibition) of the one or morehost pathways alters the level, diversity, or function of the one ormore microorganisms resident in the host in comparison to a hostorganism to which the modulating agent has not been administered. Incertain embodiments, the targeting (e.g., upregulation, downregulation,or inhibition) of the one or more host pathways increases the level,diversity, or function of the one or more microorganisms resident in thehost in comparison to a host organism to which the modulating agent hasnot been administered. In alternative embodiments, the targeting (e.g.,upregulation, downregulation, or inhibition) of the one or more hostpathways decreases the level, diversity, or function of the one or moremicroorganisms resident in the host in comparison to a host organism towhich the modulating agent has not been administered.

In some embodiments, the host pathway is a pathway that regulatesbacteriocyte function or development. In some embodiments, the targetingof bacteriocyte function or development may increase and/or decrease thelevel, diversity, and/or function of one or more microorganisms residentin the bacteriocyte in comparison to a host organism to which themodulating agent has not been administered. In certain embodiments, thetargeting of bacteriocyte function or development decreases the level,diversity, or function of one or more microorganisms resident in thebacteriocyte (e.g., a bacteriocyte of an aphid) in comparison to a hostorganism to which the modulating agent has not been administered. Incertain embodiments, the targeting of bacteriocyte function ordevelopment increases the level, diversity, or function of one or moremicroorganisms resident in the bacteriocyte (e.g., a bacteriocyte of anaphid) in comparison to a host organism to which the modulating agenthas not been administered.

In some embodiments, the host pathway is a pathway that regulates thehost's immune system. For example, in some embodiments, the modulatingagent activates an immune response against the one or moremicroorganisms resident in the host, thereby decreasing the level,diversity, and/or function of the one or more microorganisms incomparison to a host organism to which the modulating agent has not beenadministered. Alternatively, in some embodiments, the modulating agentsuppresses an immune response against the one or more microorganismsresident in the host, thereby increasing the level, diversity, and/orfunction of the one or more microorganisms in comparison to a hostorganism to which the modulating agent has not been administered.

In some embodiments, the modulating agent (e.g., a polypeptide (e.g.,antibody, bacteriocin, antimicrobial peptide, or bacteriocyte regulatorypeptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA, orinhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or a combination thereof) targetsone or more host pathways by altering gene expression in the host incomparison to a host organism to which the modulating agent has not beenadministered.

For example, the modulating agent may increase and/or decrease geneexpression in the host in comparison to a host organism to which themodulating agent has not been administered. In some embodiments, themodulating agent alters expression of a gene that encodes a proteinlisted in Table 3, Table 4, Table 5, Table 7, Table 8, or Table 9 incomparison to a host organism to which the modulating agent has not beenadministered. In some embodiments, the modulating agent decreasesexpression of a gene that encodes a protein listed in Table 3, Table 4,Table 5, Table 7, Table 8, or Table 9 in comparison to a host organismto which the modulating agent has not been administered. In someembodiments, the modulating agent decreases expression of a gene thatencodes a protein listed in Table 3, Table 4, Table 5, Table 7, Table 8,or Table 9 in comparison to a host organism to which the modulatingagent has not been administered. In some embodiments, the gene encodes abacteriocyte regulatory peptide. For example, the bacteriocyteregulatory peptide may be one listed in Table 5 or Table 8 (e.g., BCR1).In some embodiments, the gene encodes an immune system component. Forexample, the immune system component may be one listed in Table 9. Insome embodiments, the modulating agent targets a polypeptide in thehost. In some embodiments, the polypeptide is an enzyme or cellreceptor. In some embodiments, the modulating agent increases and/ordecreases enzyme activity in comparison to a host organism to which themodulating agent has not been administered. In some embodiments, themodulating agent increases and/or decreases cell receptor signaling incomparison to a host organism to which the modulating agent has not beenadministered. In some embodiments, the host protein is one listed inTable 4, Table 5, Table 8, or Table 9.

The modulating agent (e.g., a polypeptide (e.g., antibody, bacteriocin,antimicrobial peptide, or bacteriocyte regulatory peptide), a nucleicacid (e.g., DNA, RNA (e.g., mRNA, gRNA, or inhibitory RNA (e.g., RNAi,shRNA, miRNA)), CRISPR nucleic acid), a small molecule (e.g.,prostaglandin), or a combination thereof) may additionally oralternatively target one or more microbial pathways that mediateinteractions between the host and one or more microorganisms resident inthe host. In some embodiments, the modulating agent alters geneexpression in one or more microorganisms resident in the host incomparison to a host organism to which the modulating agent has not beenadministered. For example, the modulating agent may increase and/ordecrease gene expression in the one or more microorganisms in comparisonto a host organism to which the modulating agent has not beenadministered. In some embodiments, the modulating agent alters (e.g.,increases or decreases) the expression of a gene that encodes a proteinlisted in Table 3 or Table 7 in comparison to a host organism to whichthe modulating agent has not been administered. In some embodiments, themodulating agent targets (e.g., binds, antagonizes, and/or agonizes) apolypeptide in one or more microorganisms resident in the host (e.g., aprotein listed in Table 3 or Table 7).

In some embodiments, the one or more microorganisms resident in the hostis an endosymbiotic microorganism. In some embodiments, the one or moremicroorganisms is resident in the host's gut. In some embodiments, theone or more microorganisms is resident in a bacteriocyte in the host. Insome embodiments, the one or more microorganisms resident in the host isa fungus or bacterium. In some embodiments, the bacterium resident inthe host is at least one selected from the group consisting ofCandidatus spp, Buchenera spp, Blattabacterium spp, Baumania spp,Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalisspp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp,Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp,Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp,Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteriaspp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillusspp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillusspp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermusspp, Pseudomonas spp, Clostridium spp, and Escherichia spp. In someembodiments, the fungus resident in the host is at least one selectedfrom the group consisting of Candida, Metschnikowia, Debaromyces,Starmerella, Pichia, Cryptococcus, Pseudozyma, Symbiotaphrina bucneri,Symbiotaphrina Scheffersomyces shehatae, Scheffersomyces stipites,Cryptococcus, Trichosporon, Amylostereum areolatum, Epichloe spp, Pichiapinus, Hansenula capsulate, Daldinia decipien, Ceratocytis spp,Ophiostoma spp, and Attamyces bromatificus. In some embodiments, themodulating agent alters the growth, division, viability, metabolism,and/or longevity of the microorganism resident in the host. In someembodiments, the modulating agent decreases the growth, division,viability, metabolism, and/or longevity of the one or moremicroorganisms. In some embodiments, the modulating agent increases thegrowth, division, viability, metabolism, and/or longevity of the one ormore microorganisms.

In some embodiments, the modulating agent is a polypeptide (e.g.,antibody, bacteriocin, antimicrobial peptide, or bacteriocyte regulatorypeptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA, orinhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or any combination thereof.

In some embodiments, the modulating agent is a nucleic acid. The nucleicacid may be a DNA molecule, a RNA molecule (e.g., double-stranded RNA(dsRNA) or single-stranded RNA (ssRNA)), or a hybrid DNA-RNA molecule.In some embodiments, the RNA is a messenger RNA (mRNA), a guide RNA(gRNA), or an inhibitory RNA. In some embodiments, the inhibitory RNA isRNAi, shRNA, or miRNA. In some embodiments, the nucleic acid encodes apolypeptide. In some embodiments, the nucleic acid is an expressionvector encoding a polypeptide. In some embodiments, the nucleic acid isa CRISPR nucleic acid.

In some embodiments, the modulating agent is a small molecule. In someembodiments, the small molecule is an agonist, antagonist, inhibitor, oran activator of a component of a host immune system pathway orbacteriocyte regulatory pathway. In some embodiments, the small moleculeis prostaglandin.

In some embodiments, the modulating agent is a polypeptide. In someembodiments, the polypeptide is an antibody or an antibody fragment. Forexample, the antibody or antibody fragment may be an agonist orantagonist of an enzyme in the host (e.g., an immune system orbacteriocyte-regulatory enzyme) or in the microorganism resident in thehost, including any of the proteins listed in Table 5, Table 7, Table 8,or Table 9.

In some embodiments, the modulating agent (e.g., a polypeptide (e.g.,antibody, bacteriocin, antimicrobial peptide, or bacteriocyte regulatorypeptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA, orinhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or a combination thereof)modulates the host's fitness by increasing or decreasing the host'ssusceptibility to a pesticide (e.g., a pesticide listed in Table 11). Insome embodiments, the pesticide is a bactericide or fungicide. In someembodiments, the pesticide is an insecticide, molluscicide, ornematicide.

In some embodiments, the composition includes a plurality (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10) of different modulating agents(e.g., a polypeptide (e.g., antibody, bacteriocin, antimicrobialpeptide, or bacteriocyte regulatory peptide), a nucleic acid (e.g., DNA,RNA (e.g., mRNA, gRNA, or inhibitory RNA (e.g., RNAi, shRNA, miRNA)),CRISPR nucleic acid), a small molecule (e.g., prostaglandin), or acombination thereof). In some embodiments, the composition includes amodulating agent and a pesticide (e.g., a pesticide listed in Table 11).In some embodiments, the pesticide is a bactericide or fungicide. Insome embodiments, the pesticide is an insecticide, molluscicide, ornematicide. In some embodiments, the composition includes a modulatingagent and an agent that increases crop growth.

In some embodiments, the modulating agent (e.g., a polypeptide (e.g.,antibody, bacteriocin, antimicrobial peptide, or bacteriocyte regulatorypeptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA, orinhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or a combination thereof) islinked to a second moiety. In some embodiments, the second moiety isselected from the group consisting of a modulating agent, peptidenucleic acid, cell penetrating peptide (CPP), and targeting domain. Insome embodiments, the modulating agent includes a linker. In someembodiments, the linker is a cleavable linker. In some embodiments, theCPP is any one listed in Table 10.

In some embodiments, the composition further includes a carrier. In someembodiments, the carrier is an agriculturally acceptable carrier. Insome embodiments, the composition further includes a host bait, a stickyagent, or a combination thereof. In some embodiments, the host bait is acomestible agent. In some embodiments, the host bait is achemoattractant.

In some embodiments, the composition is at a dose effective to modulatehost fitness. In some embodiments, the composition is at a doseeffective to increase host fitness. In alternative embodiments, thecomposition is at a dose effective to decrease host fitness. In someembodiments, host fitness is measured by survival, lifespan,reproduction, or metabolism of the host.

In some embodiments, the composition is formulated for delivery to amicroorganism inhabiting the gut of the host. In some embodiments, thecomposition is formulated for delivery to a microorganism inhabiting abacteriocyte of the host. In some embodiments, the composition isformulated for delivery to a plant. In some embodiments, the compositionis formulated for use in a host feeding station. In some embodiments,the composition is formulated as a liquid, a powder, granules, ornanoparticles. In some embodiments, the composition is formulated as oneselected from the group consisting of a liposome, polymer, bacteriasecreting peptide, and synthetic nanocapsule. In some embodiments, thesynthetic nanocapsule delivers the composition to a target site in thehost. In some embodiments, the target site is the gut of the host. Insome embodiments, the target site is a bacteriocyte in the host.

In another aspect, provided herein are plants including any of theprevious compositions (e.g., a modulating agent (e.g., a polypeptide(e.g., antibody, bacteriocin, antimicrobial peptide, or bacteriocyteregulatory peptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA,or inhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or a combination thereof)). Insome embodiments, the plant includes a nucleic acid integrated into theplant genome, wherein the nucleic acid encodes any of the previousmodulating agents (e.g., a polypeptide (e.g., an antibody, a bacteriocin(e.g., colA), an antimicrobial peptide, a bacteriocyte regulatorypeptide, a nucleic acid, or a small molecule). The modulating agent maybe non-endogenous to the plant. In some embodiments, the plant furtherincludes a comestible agent for invertebrates (e.g., insect, mollusk, ornematode), wherein the comestible agent produces and/or carries themodulating agent. In some embodiments, the comestible agent includes oneor more components of the plant. In some embodiments, the one or morecomponents of the plant includes a root, stem, leaf, flower, sap, bark,wood, spine, pollen, nectar, seed, fruit, or any combination thereof.For example, in some embodiments, the plant produces a modulating agentthat the insect ingests by eating one or more components of the plant.

In yet another aspect, provided herein are hosts including any of theprevious compositions (e.g., a modulating agent (e.g., a polypeptide(e.g., antibody, bacteriocin, antimicrobial peptide, or bacteriocyteregulatory peptide), a nucleic acid (e.g., DNA, RNA (e.g., mRNA, gRNA,or inhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPR nucleic acid), asmall molecule (e.g., prostaglandin), or a combination thereof)). Thehost may be an invertebrate (e.g., insect, mollusk, or nematode). Insome embodiments, the invertebrate is an insect. In some embodiments,the insect is a bacteriocyte-containing insect. For example, in certainembodiments, the bacteriocyte-containing insect may be an aphid (e.g., acorn leaf aphid or green peach aphid). In further embodiments, theinsect is a beetle, weevil, fly, aphid, whitefly, leafhopper, scale,moth, butterfly, grasshopper, cricket, thrip, or mite. In otherembodiments, the invertebrate is a mollusk. In some embodiments, themollusk is a species belonging to Veronicellidae, Ampullariidae,Achatinidae, Helicidae, Hydromiidae, Planobidae, Lymnaeidae,Urocyclidae, Bradybaenidae, Agriolimacidae, Arionidae, or Milacidae. Inanother embodiment, the invertebrate may be a nematode. In someembodiments, the nematode is a species belonging to Criconematidae,Belonolaimidae, Hoplolaimidae, Heteroderidae, Longidoridae,Pratylenchidae, Trichodoridae, or Anguinidae.

In another aspect, provided herein is a system for modulating (e.g.,increasing or decreasing) a host's fitness. The system includes amodulating agent (e.g., a polypeptide (e.g., antibody, bacteriocin,antimicrobial peptide, or bacteriocyte regulatory peptide), a nucleicacid (e.g., DNA, RNA (e.g., mRNA, gRNA, or inhibitory RNA (e.g., RNAi,shRNA, miRNA)), CRISPR nucleic acid), a small molecule (e.g.,prostaglandin), or a combination thereof) that alters interactionsbetween the host and one or more microorganisms resident in the host,wherein the system is effective to modulate (e.g., increasing ordecreasing) the host's fitness, and wherein the host is an invertebrate(e.g., insect (e.g., an aphid), mollusk, or nematode). In someembodiments, the modulating agent of the system is any of the previouscompositions. In some embodiments, the modulating agent is formulated asa powder. In some embodiments, the modulating agent is formulated as asolvent. In some embodiments, the modulating agent is formulated as aconcentrate. In some embodiments, the modulating agent is formulated asa diluent. In some embodiments, the modulating agent is prepared fordelivery by combining any of the previous compositions with a carrier.

In another aspect, provided herein are methods of modulating ahost-microbiota interaction that includes delivering any of thecompositions described herein (e.g., a modulating agent (e.g., apolypeptide (e.g., antibody, bacteriocin, antimicrobial peptide, orbacteriocyte regulatory peptide), a nucleic acid (e.g., DNA, RNA (e.g.,mRNA, gRNA, or inhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPRnucleic acid), a small molecule (e.g., prostaglandin), or a combinationthereof)) to the host, wherein the modulating agent modulates one ormore interactions between the host and one or more microorganismsresident in the host.

In another aspect, provided herein are methods of modulating the fitnessof an invertebrate host (e.g., insect (e.g., an aphid), mollusk, ornematode), wherein the method includes delivering any of thecompositions described herein (e.g., a modulating agent (e.g., apolypeptide (e.g., antibody, bacteriocin, antimicrobial peptide, orbacteriocyte regulatory peptide), a nucleic acid (e.g., DNA, RNA (e.g.,mRNA, gRNA, or inhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPRnucleic acid), a small molecule (e.g., prostaglandin), or a combinationthereof)) to the host and wherein the modulating agent altersinteractions between the host and one or more microorganisms resident inthe host.

In some embodiments of any of the above methods, the one or moremicroorganisms resident in the host may be a fungus or bacterium. Insome embodiments, the one or more microorganisms is an endosymbioticmicroorganism. In some embodiments, the one or more microorganisms isresident in the host's gut. In some embodiments, the one or moremicroorganisms is resident in a bacteriocyte in the host. In someembodiments, the one or more microorganisms are required for hostfitness or host survival.

In some embodiments of any of the above methods, the modulating agentmay target one or more host pathways that mediate interactions betweenthe host and the one or more microorganisms. In some embodiments, thehost pathway is a pathway that regulates insect (e.g., an aphid)bacteriocyte function or development. In some embodiments, the targetingof the host bacteriocyte function or development decreases the level,diversity, and/or function of one or more microorganisms resident in thebacteriocyte. Alternatively, the targeting of the host bacteriocytefunction or development increases the level, diversity, and/or functionof one or more microorganisms resident in the bacteriocyte. In someembodiments, the host pathway is a pathway that regulates the host'simmune system.

In some embodiments, the modulating agent activates an immune responseagainst the one or more microorganisms resident in the host, therebydecreasing the level, diversity, and/or function of the one or moremicroorganisms. In some embodiments, the modulating agent suppresses animmune response against the one or more microorganisms resident in thehost, thereby increasing the level, diversity, and/or function of theone or more microorganisms.

In some embodiments, the modulating agent targets one or more microbialpathways that mediate interactions between the host and the one or moremicroorganisms.

In some embodiments, the delivering step includes providing themodulating agent at a dose and time sufficient to effect the one or moremicroorganisms, thereby modulating microbial diversity in the host. Insome embodiments, the delivering step includes topical application ofany of the previous compositions to a plant. In some embodiments, thedelivering step includes providing the modulating agent through agenetically modified, engineered, or transgenic plant (e.g., any of theplants described herein). In other embodiments, the delivering stepincludes providing the modulating agent to the host as a comestibleagent for invertebrates (e.g., insect, mollusk, or nematode). In furtherembodiments, the delivering step includes providing a host carrying themodulating agent. In some embodiments, the host carrying the modulatingagent can transmit the modulating agent to one or more additional hosts.

Also provided herein are screening assays to identify a modulating agentthat modulates (e.g., increases or decreases) the fitness of a host. Thescreening assay may include the steps of (a) exposing a microorganismthat can be resident in the host to one or more candidate modulatingagents and (b) identifying a modulating agent that increases ordecreases the fitness of the host. In some embodiments, the modulatingagent is a microorganism resident in the host. In some embodiments, themicroorganism is a bacterium. In some embodiments, the bacterium, whenresident in the host, increases host fitness. Alternatively, thebacterium, when resident in the host, decreases host fitness. In someembodiments, the modulating agent is any of the modulating agentsdescribed herein (e.g., a modulating agent (e.g., a polypeptide (e.g.,antibody, bacteriocin, antimicrobial peptide, or bacteriocyte regulatorypeptide (e.g., Coleoptericin A), a nucleic acid (e.g., DNA, RNA (e.g.,mRNA, gRNA, or inhibitory RNA (e.g., RNAi, shRNA, miRNA)), CRISPRnucleic acid), a small molecule (e.g., prostaglandin), or a combinationthereof)). In some embodiments, the modulating agent is provided by agenetically modified phage or bacteria. In some embodiments, the host'sfitness is modulated by modulating the host microbiota.

Definitions

As used herein, the term “bacteriocyte” refers to a specialized cellfound in invertebrates, e.g., insects, nematodes, or mollusks, whereintracellular bacteria reside with symbiotic bacterial properties. Insome instances, the bacteriocyte may be clustered with otherbacteriocytes to form a bacteriome.

As used herein, the term “effective amount” refers to an amount of amodulating agent (e.g., a polypeptide, nucleic acid, small molecule, orcombinations thereof) or composition including said agent sufficient toeffect the recited result, e.g., to increase or decrease the fitness ofa host organism (e.g., insect, nematode, or mollusk); to reach a targetlevel (e.g., a predetermined or threshold level) of a modulating agentconcentration inside a target host; to reach a target level (e.g., apredetermined or threshold level) of a modulating agent concentrationinside a target host gut; to reach a target level (e.g., a predeterminedor threshold level) of a modulating agent concentration inside a targethost bacteriocyte; to modulate the level, or an activity, of one or moremicroorganisms (e.g., endosymbiont) in the target host.

As used herein, the term “fitness” refers to the ability of a hostinvertebrate (e.g., insect, mollusk, or nematode) to survive, and/or toproduce surviving offspring. Fitness of a host (e.g., insect, mollusk,or nematode) may be measured by one or more parameters, including, butnot limited to, life span, reproductive rate, mobility, body weight, ormetabolic rate. Depending on the host, fitness may additionally bemeasured based on measures of activity (e.g., biting animals or humans,plant pollination), disease transmission (e.g., vector-vectortransmission or vector-animal transmission), or production (e.g., honeyor silk).

As used herein, the term “gut” refers to any portion of a host's gut,including, the foregut, midgut, or hindgut of the host.

As used herein, the term “host” refers to an organism, such as aninvertebrate (e.g., insect, mollusk, or nematode) carrying residentmicroorganisms (e.g., endogenous microorganisms, endosymbioticmicroorganisms (e.g., primary or secondary endosymbionts), commensalorganisms, and/or pathogenic microorganisms).

As used herein “decreasing host fitness” or “reducing host fitness”refers to any disruption to host physiology, or any activity carried outby said host, as a consequence of administration of a modulating agent,including, but not limited to, any one or more of the following desiredeffects: (1) decreasing a population of a host (e.g., insect, mollusk,or nematode) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or more; (2) decreasing the reproductive rate of a host (e.g.,insect, mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99%, 100% or more; (3) decreasing the mobility of ahost (e.g., insect, mollusk, or nematode) by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) decreasing the bodyweight of a host (e.g., insect, mollusk, or nematode) by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5)decreasing the metabolic rate or activity of a host (e.g., insect,mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 100% or more; (6) decreasing plant infestation by a host(e.g., insect, mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) decrease diseasetransmission (e.g., of a plant, animal, or human pathogen) by a host(e.g., insect, mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (8) decrease growth,increase nymphal mortality, and/or increase adult sterility of a host(e.g., insect, mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more. A decrease in host fitnesscan be determined in comparison to a host organism to which themodulating agent has not been administered.

As used herein “increasing host fitness” or “promoting host fitness”refers to any favorable alteration in host physiology, or any activitycarried out by said host, as a consequence of administration of amodulating agent, including, but not limited to, any one or more of thefollowing desired effects: (1) increasing a population of a host byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; (2) increasing the reproductive rate of a host (e.g., insect,mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 100% or more; (3) increasing the mobility of a host(e.g., insect, mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increasing the bodyweight of a host (e.g., insect, mollusk, or nematode) by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5)increasing the metabolic rate or activity of a host (e.g., insect,mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 100% or more; (6) increasing pollination (e.g., number ofplants pollinated in a given amount of time) by a host (e.g., insect,mollusk, or nematode) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 100% or more; (7) increasing production of host (e.g.,insect, mollusk, or nematode) byproducts (e.g., honey or silk) by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8)increasing nutrient content of the host (e.g., insect, mollusk, ornematode) (e.g., protein, fatty acids, or amino acids) by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (9)increasing host resistance to pesticides (e.g., insect, mollusk, ornematode) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or more. An increase in host fitness can be determined incomparison to a host organism to which the modulating agent has not beenadministered.

As used herein, “interactions between a host and microorganisms residentin the host” or “host-microbiota interactions” refer to (i) any pathways(e.g., metabolic, gene regulation, cell signaling, orimmune-inflammatory pathways) in the host that directly or indirectlyinfluences the survival, growth, or metabolism of microorganismsresident in the host (e.g., endosymbiotic microorganisms), (ii) anypathways (e.g., metabolic or cell signaling pathways) in a residentmicroorganism that directly or indirectly influences the fitness of thehost invertebrate (e.g., insect, nematode, or mollusk), and/or (iii) anypathways (e.g., metabolic, cell signaling, or immune-inflammatorypathways) in a resident microorganism that directly or indirectlyinfluences surivival, growth or metabolism of the host, and/or (iv) anypathways (e.g., metabolic, gene regulation, cell signaling, orimmune-inflammatory pathways) in the host that directly or indirectlyinfluences the fitness of the resident microorganism.

The term “insect” includes any organism belonging to the phylumArthropoda and to the class Insecta or the class Arachnida, in any stageof development, i.e., immature and adult insects.

The term “mollusk” includes any organism belonging to the phylumMollusca, including organisms of the class Gastropoda (e.g., snails andslugs), in any stage of development, i.e., immature and adult mollusks.

The term “nematode” includes any organism belonging to the phylumNematoda (e.g., nematodes) in any stage of development, i.e., immatureand adult nematodes.

As used herein, the term “microorganism” or “microbiota” refers tobacteria or fungi. Microorganisms may refer to microorganisms residentin a host organism (e.g., endogenous microorganisms, endosymbioticmicroorganisms (e.g., primary or secondary endosymbionts)) ormicroorganisms exogenous to the host, including those that may act asmodulating agents. As used herein, the term “target microorganism”refers to a microorganism that is resident in the host and impacted by amodulating agent, either directly or indirectly.

As used herein, the term “modulating agent” or “agent” refers to anagent that is capable of altering the levels and/or functioning ofmicroorganisms resident in a host organism (e.g., invertebrate, e.g.,insect, mollusk, or nematode), and thereby modulate (e.g., increase ordecrease) the fitness of the host organism.

As defined herein, the term “nucleic acid” and “polynucleotide” areinterchangeable and refer to RNA or DNA that is linear or branched,single or double stranded, or a hybrid thereof, regardless of length(e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150,200, 250, 500, 1000, or more nucleic acids). The term also encompassesRNA/DNA hybrids. Nucleotides are typically linked in a nucleic acid byphosphodiester bonds, although the term “nucleic acid” also encompassesnucleic acid analogs having other types of linkages or backbones (e.g.,phosphoramide, phosphorothioate, phosphorodithioate,O-methylphosphoroamidate, morpholino, locked nucleic acid (LNA),glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptidenucleic acid (PNA) linkages or backbones, among others). The nucleicacids may be single-stranded, double-stranded, or contain portions ofboth single-stranded and double-stranded sequence. A nucleic acid cancontain any combination of deoxyribonucleotides and ribonucleotides, aswell as any combination of bases, including, for example, adenine,thymine, cytosine, guanine, uracil, and modified or non-canonical bases(including, e.g., hypoxanthine, xanthine, 7-methylguanine,5,6-dihydrouracil, 5-methylcytosine, and 5 hydroxymethylcytosine).

As used herein, the term “pest” refers to invertebrates (e.g., insects,nematodes, or mollusks) that cause damage to plants or other organisms,or otherwise are detrimental to humans, for example, human agriculturalmethods or products.

As used herein, the term “pesticide” or “pesticidal agent” refers to asubstance that can be used in the control of agricultural,environmental, and domestic/household pests, such as insects, mollusks,nematodes, fungi, bacteria, and viruses. The term “pesticide” isunderstood to encompass naturally occurring or synthetic insecticides(larvicides or adulticides), insect growth regulators, nematicides,molluscicides, acaricides (miticides), nematicides, ectoparasiticides,bactericides, fungicides, or herbicides (substance which can be used inagriculture to control or modify plant growth). Further examples ofpesticides or pesticidal agents are listed in Table 11. In someinstances, the pesticide is an allelochemical. As used herein,“allelochemical” or “allelochemical agent” is a substance produced by anorganism that can effect a physiological function (e.g., thegermination, growth, survival, or reproduction) of another organism(e.g., an insect, mollusk, or nematode).

As used herein, the term “peptide,” “protein,” or “polypeptide”encompasses any chain of naturally or non-naturally occurring aminoacids (either D- or L-amino acids), regardless of length (e.g., at least2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or moreamino acids), the presence or absence of post-translationalmodifications (e.g., glycosylation or phosphorylation), or the presenceof, e.g., one or more non-amino acyl groups (for example, sugar, lipid,etc.) covalently linked to the peptide, and includes, for example,natural proteins, synthetic, or recombinant polypeptides and peptides,hybrid molecules, peptoids, or peptidomimetics.

As used herein, “percent identity” between two sequences is determinedby the BLAST 2.0 algorithm, which is described in Altschul et al., J.Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

As used herein, the term “plant” refers to whole plants, plant organs,plant tissues, seeds, plant cells, seeds, or progeny of the same. Plantcells include, without limitation, cells from seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen, or microspores. Plant partsinclude differentiated and undifferentiated tissues including, but notlimited to the following: roots, stems, shoots, leaves, pollen, seeds,tumor tissue, or various forms of cells and culture (e.g., single cells,protoplasts, embryos, and callus tissue). The plant tissue may be in aplant or in a plant organ, tissue, or cell culture.

As used herein a “transgenic plant,” “genetically engineered plant,” or“genetically modified plant” refers to a plant whose genome (e.g.,chromosomal DNA, chloroplast DNA, or mitochondrial DNA) has been alteredby the stable integration of recombinant DNA. A transgenic plantincludes a plant regenerated from an originally-transformed plant celland progeny transgenic plants from later generations or crosses of atransformed plant. For example, a transgenic plant may be geneticallyengineered to produce a heterologously (e.g., non-endogenous) ornon-heterologously (e.g., endogenous) encoded protein or RNA, forexample, of any of the modulating agents in the methods or compositionsdescribed herein. Any plant species may be transformed to create atransgenic plant. The transgenic plant may be a dicotyledonous plant ora monocotyledonous plant. For example and without limitation, transgenicplants of the compositions and methods described herein may be derivedfrom any of the following diclotyledonous plant families: Leguminosae,including plants such as pea, alfalfa and soybean; Umbelliferae,including plants such as carrot and celery; Solanaceae, including theplants such as tomato, potato, aubergine, tobacco, and pepper;Cruciferae, particularly the genus Brassica, which includes plant suchas oilseed rape, beet, cabbage, cauliflower and broccoli); andArabidopsis thaliana; Compositae, which includes plants such as lettuce;Malvaceae, which includes cotton; Fabaceae, which includes plants suchas peanut, and the like. Transgenic plants of the invention may bederived from monocotyle-donous plants, such as, for example, wheat,barley, sorghum, millet, rye, triticale, maize, rice, oats, switchgrass,miscanthus, and sugarcane. Transgenic plants of the invention are alsoembodied as trees such as apple, pear, quince, plum, cherry, peach,nectarine, apricot, papaya, mango, and other woody species includingconiferous and deciduous trees such as poplar, pine, sequoia, cedar,oak, willow, and the like.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description and the Claims.

BRIEF DESCRIPTION OF THE FIGURES

The figures are meant to be illustrative of one or more features,aspects, or embodiments of the invention and are not intended to belimiting.

FIG. 1 is a panel of graphs showing that treatment with P. pastorisdelayed aphid development. First and second instar LSR-1 aphids wereplaced on leaves perfused with water (negative control) or with asolution of P. pastoris in water and developmental stage was monitoredat each indicated time point during the experiment. Shown are the meanpercentages of aphids in each group±SD.

FIG. 2 is a graph showing that P. pastoris treatment resulted in aphiddeath. First and second instar LSR-1 aphids were treated with water(control) or with P. pastoris via leaf perfusion and survival wasmonitored daily during the experiment. N=62-63 aphids/group.Statistically significant differences were determined using Log-Rank(Mantel Cox) test. ****, p<0.0001.

FIG. 3 is a panel of graphs showing that P. pastoris treatment via leafspraying did not affect aphid development. First and second instar LSR-1aphids were treated with water (control) or P. pastoris via airbrushspraying and developmental stage was monitored over time. Shown are themean percentages±SD of dead or live aphids in each developmental stage(1st, 2nd, 3rd, 4th, or 5th instar) at each time point. N=two replicatesof 30 aphids/group.

FIG. 4 is a graph showing that P. pastoris treatment increased aphidmortality. First and second instar LSR-1 aphids were placed on leavessprayed with water (control) or P. pastoris and survival was monitoredover time. N=60 aphids/treatment group.

FIGS. 5A and 5B are graphs showing that spraying P. pastoris on favabean leaves reduced endosymbiotic Buchnera in aphids feeding on theleaves. Symbiont titer was determined at 6 (A) and 9 (B) dayspost-treatment with P. pastoris. Shown are the mean Buchnera/aphidcopies±SD. The number in the box above the indicated dataset representsthe median value of that group. Each dot represents a single aphid.

FIG. 6 is a graph showing microinjection of BCR-4 PNA reduced BCR-4expression. Fourth and fifth instar A. pisum aphids were injected with20 nl water or 321 ng/ul of BCR-4 PNA, RNA was extracted from aphidsafter 7 days, and RT-qPCR was performed to measure expression of BCR-4.Shown are the mean BCR-4/Actin copies±SD. Each data point represents asingle aphid. The number in the box above each dataset represents themedian of the data.

FIG. 7 is a graph showing the decrease in insect survival aftertreatment with a PNA to BCR-4. Fourth and fifth instar A. pisum aphidswere injected with water or with a PNA to BCR-4. Survival was monitoreddaily over the course of the experiment. N=20 aphids per treatmentgroup.

FIG. 8 is a graph showing injection of aphids with a PNA to BCR-4resulted in decreased fecundity. Fourth and fifth instar A. pisum aphidswere injected with water or with a PNA to BCR-4. Fecundity was measuredby counting the number of offspring produced by each group at each timepoint and is represented as the number of F1's (first generationoffspring) produced by F0 (adults) per day. N=20 aphids per treatmentgroup.

FIG. 9 is a panel of graphs showing treatment with BCR-4 PNA delayedaphid development. First and second instar LSR-1 aphids were placed onleaves perfused with water (negative control) or with a solution ofBCR-4 PNA in water and developmental stage was monitored at eachindicated time point during the experiment. Shown are the meanpercentages of aphids in each group±SD.

FIG. 10 is a graph showing BCR-4 treatment resulted in increased aphiddeath. First and second instar LSR-1 aphids were treated with water(control) or with BCR-4 PNA via leaf perfusion and survival wasmonitored daily during the experiment. N=60 aphids/group. Statisticallysignificant differences were determined using Log-Rank (Mantel Cox)test.

FIG. 11 is a graph showing treatment with BCR-4 PNA delivered via leafperfusion increased Buchnera titers. First and second instar LSR-1aphids were treated with water (control) or BCR4 PNA via leaf perfusionand dead aphids were collected on days 5 and 6 after treatment. DNA wasextracted, and qPCR was performed to determine the number ofBuchnera/aphid DNA copies. Shown are the mean number of Buchnera/aphidDNA copies±SD of 6-7 aphids/group.

FIG. 12 is a graph showing treatment of aphids with a PNA against BCR-4via leaf perfusion resulted in a reduction of BCR-4 expression. Firstand second instar LSR-1 aphids were treated with water (control) orBCR-4 PNA via leaf perfusion and on day 7, RNA was extracted from livingaphids and RT-qPCR was performed to quantify expression of BCR-4relative to actin expression. The number in the box represents themedian of the dataset.

FIG. 13 is a graph showing treatment with dsRNA-ApGLNT1 knocked down theexpression of ApGLNT1. Fifth instar A. pisum aphids were injected withwater or dsRNA-ApGLNT1 in water. At 2 days post-treatment, total RNA wasextracted and RT-qPCR was performed to determine ApGLNT1 gene relativeexpression (Actin as internal reference gene). Shown is the mean ratioof relative expression of ApGLNT1/Actin±SD of 5-7 aphids/group.Statistically significant differences were determined by Student'sT-test (**, p<0.01).

FIG. 14 is a graph showing treatment with dsRNA-ApGLNT1 increased aphidmortality. LSR-1 A. pisum aphids were injected with water ordsRNA-ApGLNT1 in water and survival was monitored over the course of theexperiment. N=40 aphids/group. A statistically significant differencewas identified between the two groups as determined using a Log-Rank(Mantel Cox) test.

FIG. 15 is a graph showing treatment with dsRNA-ApGLNT1 resulted indecreased Buchnera titers. LSR-1 A. pisum aphids were injected withwater or dsRNA-ApGLNT1 in water, DNA was extracted from aphids at 5 dayspost-injection, and qPCR was performed to quantify Buchnera. Shown arethe mean copies of Buchnera/aphid DNA±SD. Each dot represents anindividual aphid. The number in the box above each data set representsthe median of the group.

FIGS. 16A and 16B are a panel of graphs showing offspring from aphidsmicroinjected with dsRNA-ApGLNT1 displayed delayed development. FIG.16A: LSR-1 A. pisum aphids were injected with water or dsRNA-ApGLNT1 inwater and life stages were monitored at the indicated time point afterinjection. Shown is the mean percent±of aphids that were dead or aliveat each instar stage throughout the experiment. N=40 aphids/group. FIG.16B: 4 days after offspring were collected, images were taken of eachaphid to determine the area of each aphid. Shown is the mean area±SD ofoffspring taken from aphids injected with either water or dsRNA-ApGLNT1.Statistically significant differences were determined using a Student'st-test. Each data point represents one individual aphid.

FIG. 17 is an illustration showing the dsRNA expression cassette. pCaMV35S promoter is placed upstream of the dsRNA expressing sequence. Thesense and the antisense strands of a region of the target aphid gene areplaced in tandem with a small spacer which will act as the hairpin loop.Once expressed, the RNA formed will assume a double strandedconfiguration due to the complementarity of the sequence.

FIG. 18 is an illustration of the shuttle vector for the constructs forexpressing dsRNA in N. tabacum. The plasmid includes origins ofreplications compatible with E. coli and A. tumefaciens, kanamycin andgentamycin resistance markers, green fluorescence expression cassetteunder a parsley ubiquitin promoter, and finally the dsRNA expressioncassette driven by the pCaMV 35S.

FIG. 19 is a panel of images showing GFP expression in N. tabacum plantinfiltrated by A. tumefaciens. The top panels are N. tabacum infiltratedwith A. tumefaciens containing a plasmid that can constitutively drivethe expression of GFP in N. tabacum (Top left is brightfield, and topright is green channel). The bottom panels are negative control leavesnot infiltrated by A. tumefaciens.

DETAILED DESCRIPTION

Provided herein are methods and compositions for modulating the fitnessof a host invertebrate (e.g., insect, mollusk, or nematode) by alteringinteractions between the host and one or more microorganisms resident inthe host. The invention features a composition including a modulatingagent (e.g., a polypeptide, nucleic acid, small molecule, orcombinations thereof) that can indirectly induce changes in the host'smicrobiota in a manner that modulates (e.g., increases or decreases)host fitness. For example, the modulating agent may target host pathways(e.g., immune system or bacteriocyte pathways) or microbial pathwaysthat alter (e.g., increase or decrease) microbial levels, microbialactivity, microbial metabolism, and/or microbial diversity, and in turnmodulates (e.g., increase or decrease) the fitness of a variety ofinvertebrates (e.g., insect, mollusk, or nematode) that are importantfor agriculture, commerce, and/or public health.

The methods and compositions described herein are based, in part, on theexamples which illustrate how different agents, for example, smallcompounds (e.g., prostaglandin), inhibitory RNA (e.g., dsRNA or PNAs),and microorganisms (fungi or bacteria) can be used to alter the host'simmune system response towards microorganisms resident in the host. Themethods and compositions described herein can also be used to alter thefunction of host organs or cells in which microorganisms typicallyreside. For example, RNA may be used to impair bacteriocyte function inan aphid, thereby disrupting endosymbiotic microorganism populationsresident in the bacteriocyte of the aphid. Disruption of endosymbioticpopulations of microorganisms (e.g., Buchnera spp.) in the aphid, inturn, decreases the fitness of the aphid. Nucleic acids, such as RNAs(e.g., dsRNA) or PNAs, or small molecules (e.g., prostaglandin) arerepresentative of modulating agents useful in the invention, and othermodulating agents of this type may be useful in the invention. On thisbasis, the present disclosure describes a variety of differentapproaches for the use of agents that modulates (e.g., increases ordecreases) the fitness of an invertebrate host (e.g., insect, mollusk,or nematode), wherein the modulating agent alters interactions betweenthe host and one or more microorganisms resident in the host.

I. Hosts

i. Insect Hosts

In some instances, the host described herein is an organism belonging tothe phylum Arthropoda. In some instances, the insect is considered apest, e.g., an agricultural pest. In some instances, the insect carriesa bacterium or virus that is considered a plant pest that causes diseasein a plant (e.g., Agrobacterium or tomato yellow leaf curl virus(TYLCV)). The host may be at any stage developmentally. For instance,the host may be an embryo, a larva, a pupa, or an adult.

In some instances, the insect may belong to the following orders: Acari,Araneae, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera,Diplura, Diptera (e.g., spotted-wing Drosophila), Embioptera,Ephemeroptera, Grylloblatodea, Hemiptera (e.g., an aphid, Greenhouswhitefly, or stinkbug), Homoptera, Hymenoptera, Isoptera, Lepidoptera,Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phasmida,Plecoptera, Protura, Psocoptera, Siphonaptera, Siphunculata, Thysanura,Strepsiptera, Thysanoptera, Trichoptera, or Zoraptera.

In some instances, the insect is from the class Arachnida, for example,Acarus spp., Aceria sheldoni, Aculops spp., Aculus spp., Amblyomma spp.,Amphitetranychus viennensis, Argas spp., Boophilus spp., Brevipalpusspp., Bryobia graminum, Bryobia praetiosa, Centruroides spp., Chorioptesspp., Dermanyssus gallinae, Dermatophagoides pteronyssinus,Dermatophagoides farinae, Dermacentor spp., Eotetranychus spp.,Epitrimerus pyri, Eutetranychus spp., Eriophyes spp., Glycyphagusdomesticus, Halotydeus destructor, Hemitarsonemus spp., Hyalomma spp.,Ixodes spp., Latrodectus spp., Loxosceles spp., Metatetranychus spp.,Neutrombicula autumnalis, Nuphersa spp., Oligonychus spp., Ornithodorusspp., Ornithonyssus spp., Panonychus spp., Phyllocoptruta oleivora,Polyphagotarsonemus latus, Psoroptes spp., Rhipicephalus spp.,Rhizoglyphus spp., Sarcoptes spp., Scorpio maurus, Steneotarsonemusspp., Steneotarsonemus spinki, Tarsonemus spp., Tetranychus spp.,Trombicula alfreddugesi, Vaejovis spp., Vasates lycopersici.

In some instances, the insect is from the class Chilopoda, for example,Geophilus spp., Scutigera spp.

In some instances, the insect is from the order Collembola, for example,Onychiurus armatus.

In some instances, the insect is from the class Diplopoda, for example,Blaniulus guttulatus.

In some instances, the insect is from the class Insecta, e.g. from theorder Blattodea, for example, Blattella asahinai, Blattella germanica,Blatta orientalis, Leucophaea maderae, Panchlora spp., Parcoblatta spp.,Periplaneta spp., Supella longipalpa.

In some instances, the insect is from the order Coleoptera, for example,Acalymma vittatum, Acanthoscelides obtectus, Adoretus spp., Agelasticaalni, Agriotes spp., Alphitobius diaperinus, Amphimallon solstitialis,Anobium punctatum, Anoplophora spp., Anthonomus spp., Anthrenus spp.,Apion spp., Apogonia spp., Atomaria spp., Attagenus spp., Bruchidiusobtectus, Bruchus spp., Cassida spp., Cerotoma trifurcata,Ceutorrhynchus spp., Chaetocnema spp., Cleonus mendicus, Conoderus spp.,Cosmopolites spp., Costelytra zealandica, Ctenicera spp., Curculio spp.,Cryptolestes ferrugineus, Cryptorhynchus lapathi, Cylindrocopturus spp.,Dendroctonus spp. (e.g., Dendroctonus ponderosae), Dermestes spp.,Diabrotica spp. (e.g., corn rootworm), Dichocrocis spp., Dicladispaarmigera, Diloboderus spp., Epilachna spp., Epitrix spp., Faustinusspp., Gibbium psylloides, Gnathocerus cornutus, Hellula undalis,Heteronychus arator, Heteronyx spp., Hylamorpha elegans, Hylotrupesbajulus, Hypera postica, Hypomeces squamosus, Hypothenemus spp.(Hypothenemus hampei), Lachnosterna consanguinea, Lasioderma serricorne,Latheticus oryzae, Lathridius spp., Lema spp., Leptinotarsadecemlineata, Leucoptera spp., Lissorhoptrus oryzophilus, Lixus spp.,Luperodes spp., Lyctus spp., Megascelis spp., Melanotus spp., Meligethesaeneus, Melolontha spp., Migdolus spp., Monochamus spp., Naupactusxanthographus, Necrobia spp., Niptus hololeucus, Oryctes rhinoceros,Oryzaephilus surinamensis, Oryzaphagus oryzae, Otiorrhynchus spp.,Oxycetonia jucunda, Phaedon cochleariae, Phyllophaga spp., Phyllophagahelleri, Phyllotreta spp., Popillia japonica, Premnotrypes spp.,Prostephanus truncatus, Psyffiodes spp., Ptinus spp., Rhizobiusventralis, Rhizopertha dominica, Sitophilus spp., Sitophilus oryzae,Sphenophorus spp., Stegobium paniceum, Sternechus spp., Symphyletesspp., Tanymecus spp., Tenebrio molitor, Tenebrioides mauretanicus,Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus spp., Zabrusspp.;

from the order Diptera, for example, Aedes spp., Agromyza spp.,Anastrepha spp., Anopheles spp., Asphondylia spp., Bactrocera spp.,Bibio hortulanus, Calliphora erythrocephala, Calliphora vicina,Ceratitis capitata, Chironomus spp., Chrysomyia spp., Chrysops spp.,Chrysozona pluvialis, Cochliomyia spp., Contarinia spp., Cordylobiaanthropophaga, Cricotopus sylvestris, Culex spp., Culicoides spp.,Culiseta spp., Cuterebra spp., Dacus oleae, Dasyneura spp., Delia spp.,Dermatobia hominis, Drosophila spp., Echinocnemus spp., Fannia spp.,Gasterophilus spp., Glossina spp., Haematopota spp., Hydrellia spp.,Hydrellia griseola, Hylemya spp., Hippobosca spp., Hypoderma spp.,Liriomyza spp., Lucilia spp., Lutzomyia spp., Mansonia spp., Musca spp.(e.g., Musca domestica), Oestrus spp., Oscinella frit, Paratanytarsusspp., Paralauterborniella subcincta, Pegomyia spp., Phlebotomus spp.,Phorbia spp., Phormia spp., Piophila casei, Prodiplosis spp., Psilarosae, Rhagoletis spp., Sarcophaga spp., Simulium spp., Stomoxys spp.,Tabanus spp., Tetanops spp., Tipula spp.

In some instances, the insect is from the order Heteroptera, forexample, Anasa tristis, Antestiopsis spp., Boisea spp., Blissus spp.,Calocoris spp., Campylomma livida, Cavelerius spp., Cimex spp., Collariaspp., Creontiades dilutus, Dasynus piperis, Dichelops furcatus,Diconocoris hewetti, Dysdercus spp., Euschistus spp., Eurygaster spp.,Heliopeltis spp., Horcias nobilellus, Leptocorisa spp., Leptocorisavaricornis, Leptoglossus phyllopus, Lygus spp., Macropes excavatus,Miridae, Monalonion atratum, Nezara spp., Oebalus spp., Pentomidae,Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea,Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea,Scotinophora spp., Stephanitis nashi, Tibraca spp., Triatoma spp.

In some instances, the insect is from the order Hemiptera or suborderHomoptera, for example, Acizzia acaciaebaileyanae, Acizzia dodonaeae,Acizzia uncatoides, Acrida turrita, Acyrthosipon spp., Acrogonia spp.,Aeneolamia spp., Agonoscena spp., Aleyrodes proletella, Aleurolobusbarodensis, Aleurothrixus floccosus, Allocaridara malayensis, Amrascaspp., Anuraphis cardui, Aonidiella spp., Aphanostigma pini, Aphis spp.(e.g., Apis gossypii), Arboridia apicalis, Arytainilla spp., Aspidiellaspp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia tabaci,Blastopsylla occidentalis, Boreioglycaspis melaleucae, Brachycaudushelichrysi, Brachycolus spp., Brevicoryne brassicae, Cacopsylla spp.,Calligypona marginata, Carneocephala fulgida, Ceratovacuna lanigera,Cercopidae, Ceroplastes spp., Chaetosiphon fragaefolii, Chionaspistegalensis, Chlorita onukii Chondracris rosea, Chromaphis juglandicola,Chrysomphalus ficus, Cicadulina mbila, Coccomytilus haffi, Coccus spp.,Cryptomyzus ribis, Cryptoneossa spp., Ctenarytaina spp., Dalbulus spp.,Dialeurodes citri, Diaphorina citri, Diaspis spp., Drosicha spp.,Dysaphis spp., Dysmicoccus spp., Empoasca spp., Eriosoma spp.,Erythroneura spp., Eucalyptolyma spp., Euphyllura spp., Euscelisbilobatus, Ferrisia spp., Geococcus coffeae, Glycaspis spp.,Heteropsylla cubana, Heteropsylla spinulosa, Homalodisca coagulata,Homalodisca vitripennis, Hyalopterus arundinis, Icerya spp., Idiocerusspp., Idioscopus spp., Laodelphax striatellus, Lecanium spp.,Lepidosaphes spp., Lipaphis erysimi, Macrosiphum spp., Macrostelesfacifrons, Mahanarva spp., Melanaphis sacchari, Metcalfiella spp.,Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, Myzusspp., Nasonovia ribisnigri, Nephotettix spp., Nettigoniclla spectra,Nilaparvata lugens, Oncometopia spp., Orthezia praelonga, Oxyachinensis, Pachypsylla spp., Parabemisia myricae, Paratrioza spp.,Parlatoria spp., Pemphigus spp., Peregrinus maidis, Phenacoccus spp.,Phloeomyzus passerinii, Phorodon humuli, Phylloxera spp., Pinnaspisaspidistrae, Planococcus spp., Prosopidopsylla flava, Protopulvinariapyriformis, Pseudaulacaspis pentagona, Pseudococcus spp., Psyllopsisspp., Psylla spp., Pteromalus spp., Pyrilla spp., Quadraspidiotus spp.,Quesada gigas, Rastrococcus spp., Rhopalosiphum spp., Saissetia spp.,Scaphoideus titanus, Schizaphis graminum, Selenaspidus articulatus,Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina,siphoninus phillyreae, Tenalaphara malayensis, Tetragonocephela spp.,Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodesvaporariorum, Trioza spp., Typhlocyba spp., Unaspis spp., Viteusvitifolii, Zygina spp.; from the order Hymenoptera, for example,Acromyrmex spp., Athalia spp., Atta spp., Diprion spp., Hoplocampa spp.,Lasius spp., Monomorium pharaonis, Sirex spp., Solenopsis invicta,Tapinoma spp., Urocerus spp., Vespa spp., Xeris spp. In certaininstances, the insect is an aphid (e.g., Rhopalosiphum maidis or Myzuspersicae).

In some instances, the insect is from the order Isopoda, for example,Armadillidium vulgare, Oniscus asellus, Porcellio scaber.

In some instances, the insect is from the order Isoptera, for example,Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermesspp., Microtermes obesi, Odontotermes spp., Reticulitermes spp.

In some instances, the insect is from the order Lepidoptera, forexample, Achroia grisella, Acronicta major, Adoxophyes spp., Aedialeucomelas, Agrotis spp., Alabama spp., Amyelois transitella, Anarsiaspp., Anticarsia spp., Argyroploce spp., Barathra brassicae, Borbocinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola spp.,Cacoecia spp., Caloptilia theivora, Capua reticulana, Carpocapsapomonella, Carposina niponensis, Cheimatobia brumata, Chilo spp.,Choristoneura spp., Clysia ambiguella, Cnaphalocerus spp.,Cnaphalocrocis medinalis, Cnephasia spp., Conopomorpha spp.,Conotrachelus spp., Copitarsia spp., Cydia spp., Dalaca noctuides,Diaphania spp., Diatraea saccharalis, Earias spp., Ecdytolophaaurantium, Elasmopalpus lignosellus, Eldana saccharina, Ephestia spp.,Epinotia spp., Epiphyas postvittana, Etiella spp., Eulia spp.,Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Feltia spp., Galleriamellonella, Gracillaria spp., Grapholitha spp., Hedylepta spp.,Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella,Homoeosoma spp., Homona spp., Hyponomeuta padella, Kakivoriaflavofasciata, Laphygma spp., Laspeyresia molesta, Leucinodes orbonalis,Leucoptera spp., Lithocolletis spp., Lithophane antennata, Lobesia spp.,Loxagrotis albicosta, Lymantria spp., Lyonetia spp., Malacosomaneustria, Maruca testulalis, Mamstra brassicae, Melanitis leda, Mocisspp., Monopis obviella, Mythimna separata, Nemapogon cloacellus,Nymphula spp., Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp.,Oulema oryzae, Panolis flammea, Parnara spp., Pectinophora spp.,Perileucoptera spp., Phthorimaea spp., Phyllocnistis citrella,Phyllonorycter spp., Pieris spp., Platynota stultana, Plodiainterpunctella, Plusia spp., Plutella xylostella, Prays spp., Prodeniaspp., Protoparce spp., Pseudaletia spp., Pseudaletia unipuncta,Pseudoplusia includens, Pyrausta nubilalis, Rachiplusia nu, Schoenobiusspp., Scirpophaga spp., Scirpophaga innotata, Scotia segetum, Sesamiaspp., Sesamia inferens, Sparganothis spp., Spodoptera spp., Spodopterapraefica, Stathmopoda spp., Stomopteryx subsecivella, Synanthedon spp.,Tecia solanivora, Thermesia gemmatalis, Tinea cloacella, Tineapellionella, Tineola bisselliella, Tortrix spp., Trichophaga tapetzella,Trichoplusia spp., Tryporyza incertulas, Tuta absoluta, Virachola spp.

In some instances, the insect is from the order Orthoptera orSaltatoria, for example, Acheta domesticus, Dichroplus spp., Gryllotalpaspp., Hieroglyphus spp., Locusta spp., Melanoplus spp., Schistocercagregaria.

In some instances, the insect is from the order Phthiraptera, forexample, Damalinia spp., Haematopinus spp., Linognathus spp., Pediculusspp., Ptirus pubis, Trichodectes spp.

In some instances, the insect is from the order Psocoptera for exampleLepinatus spp., Liposcells spp.

In some instances, the insect is from the order Siphonaptera, forexample, Ceratophyllus spp., Ctenocephalides spp., Pulex irritans, Tungapenetrans, Xenopsylla cheopsis.

In some instances, the insect is from the order Thysanoptera, forexample, Anaphothrips obscurus, Baliothrips biformis, Drepanothripsreuteri, Enneothrips flavens, Frankliniella spp., Hellothrips spp.,Hercinothrips femoralis, Rhipiphorothrips cruentatus, Scirtothrips spp.,Taeniothrips cardamomi, Thrips spp.

In some instances, the insect is from the order Zygentoma (=Thysanura),for example, Ctenolepisma spp., Lepisma saccharina, Lepismodesinquilinus, Thermobia domestica.

In some instances, the insect is from the class Symphyla, for example,Scutigerella spp.

In some instances, the insect is a mite, including but not limited to,Tarsonemid mites, such as Phytonemus pallidus, Polyphagotarsonemuslatus, Tarsonemus bilobatus, or the like; Eupodid mites, such asPenthaleus erythrocephalus, Penthaleus major, or the like; Spider mites,such as Oligonychus shinkajii, Panonychus citri, Panonychus mori,Panonychus ulmi, Tetranychus kanzawai, Tetranychus urticae, or the like;Eriophyid mites, such as Acaphylla theavagrans, Aceria tulipae, Aculopslycopersici, Aculops pelekassi, Aculus schlechtendali, Eriophyeschibaensis, Phyllocoptruta oleivora, or the like; Acarid mites, such asRhizoglyphus robini, Tyrophagus putrescentiae, Tyrophagus similis, orthe like; Bee brood mites, such as Varroa jacobsoni, Varroa destructoror the like; Ixodides, such as Boophilus microplus, Rhipicephalussanguineus, Haemaphysalis longicornis, Haemophysalis flava,Haemophysalis campanulata, Ixodes ovatus, Ixodes persulcatus, Amblyommaspp., Dermacentor spp., or the like; Cheyletidae, such as Cheyletiellayasguri, Cheyletiella blakei, or the like; Demodicidae, such as Demodexcanis, Demodex cati, or the like; Psoroptidae, such as Psoroptes ovis,or the like; Scarcoptidae, such as Sarcoptes scabiei, Notoedres cati,Knemidocoptes spp., or the like.

The methods and compositions provided herein may be used with any insecthost that is considered a vector for a pathogen that is capable ofcausing disease in animals. For example, the insect host may include,but is not limited to those with piercing-sucking mouthparts, as foundin Hemiptera and some Hymenoptera and Diptera such as mosquitoes, bees,wasps, midges, lice, tsetse fly, fleas and ants, as well as members ofthe Arachnidae such as ticks and mites; order, class or family ofAcarina (ticks and mites) e.g. representatives of the familiesArgasidae, Dermanyssidae, Ixodidae, Psoroptidae or Sarcoptidae andrepresentatives of the species Amblyomma spp., Anocenton spp., Argasspp., Boophilus spp., Cheyletiella spp., Chorioptes spp., Demodex spp.,Dermacentor spp., Denmanyssus spp., Haemophysalis spp., Hyalomma spp.,Ixodes spp., Lynxacarus spp., Mesostigmata spp., Notoednes spp.,Ornithodoros spp., Ornithonyssus spp., Otobius spp., otodectes spp.,Pneumonyssus spp., Psoroptes spp., Rhipicephalus spp., Sancoptes spp.,or Trombicula spp.; Anoplura (sucking and biting lice) e.g.representatives of the species Bovicola spp., Haematopinus spp.,Linognathus spp., Menopon spp., Pediculus spp., Pemphigus spp.,Phylloxera spp., or Solenopotes spp.; Diptera (flies) e.g.representatives of the species Aedes spp., Anopheles spp., Calliphoraspp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Cw/ex spp.,Culicoides spp., Cuterebra spp., Dermatobia spp., Gastrophilus spp.,Glossina spp., Haematobia spp., Haematopota spp., Hippobosca spp.,Hypoderma spp., Lucilia spp., Lyperosia spp., Melophagus spp., Oestrusspp., Phaenicia spp., Phlebotomus spp., Phormia spp., Sarcophaga spp.,Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp. or Zzpu/alphaspp.; Mallophaga (biting lice) e.g. representatives of the speciesDamalina spp., Felicola spp., Heterodoxus spp. or Trichodectes spp.; orSiphonaptera (wingless insects) e.g. representatives of the speciesCeratophyllus spp., Xenopsylla spp; Cimicidae (true bugs) e.g.representatives of the species Cimex spp., Tritominae spp., Rhodiniusspp., or Triatoma spp. In some instances, the insect is a blood-suckinginsect from the order Diptera (e.g., suborder Nematocera, e.g., familyColicidae). In some instances, the insect is from the subfamiliesCulicinae, Corethrinae, Ceratopogonidae, or Simuliidae. In someinstances, the insect is of a Culex spp., Theobaldia spp., Aedes spp.,Anopheles spp., Aedes spp., Forciponiyia spp., Culicoides spp., or Heleaspp.

ii. Mollusk Hosts

In some instances, the host described herein may be an organismbelonging to the phylum Mollusca. In some instances, the mollusk isconsidered a pest, e.g., an agricultural pest. For example, the methodsand compositions are suitable for controlling terrestrial Gastropods(e.g., slugs and snails) in agriculture and horticulture. They includeall terrestrial slugs and snails which mostly occur as polyphagous pestson agricultural and horticultural crops.

In some instances, the mollusk belongs to the family Achatinidae,Agriolimacidae, Ampullariidae, Arionidae, Bradybaenidae, Helicidae,Hydromiidae, Lymnaeidae, Milacidae, Urocyclidae, or Veronicellidae.

For example, in some instances, the mollusk is Achatina spp., Agriolimaxspp., Arion spp. (e.g., A. ater, A. circumscriptus, A. distinctus, A.fasciatus, A. hortensis, A. intermedius, A. rufus, A. subfuscus, A.silvaticus, A. lusitanicus), Biomphalaria spp., Bradybaena spp. (e.g.,B. fruticum), Bulinus spp., Cantareus spp. (e.g., C. asperses), Cepaeaspp. (e.g., C. hortensis, C. nemoralis), Cernuella spp., Cochlicellaspp., Cochlodina spp. (e.g., C. laminata), Deroceras spp. (e.g., D.agrestis, D. empiricorum, D. laeve, D. panornimatum, D. reticulatum),Discus spp. (e.g., D. rotundatus), Euomphalia spp., Galba spp. (e.g., G.trunculata), Helicella spp. (e.g., H. itala, H. obvia), Helicigona spp.(e.g., H. arbustorum), Helicodiscus spp., Helix spp. (e.g., H. aperta,H. aspersa, H. pomatia), Limax spp. (e.g., L. cinereoniger, L. flavus,L. marginatus, L. maximus, L. tenellus), Lymnaea spp. (e.g., L.stagnalis), Milax spp. (e.g., M. gagates, M. marginatus, M. sowerbyi, M.budapestensis), Oncomelania spp., Opeas spp., Oxyloma spp. (e.g., O.pfeifferi), Pomacea spp. (e.g., P. canaliculata), Succinea spp.,Tandonia spp. (e.g., T. budapestensis, T. sowerbyi), Theba spp.,Vallonia spp., and Zonitoides spp. (e.g., Z. nitidus).

iii. Nematode Hosts

The host of any of the compositions or methods described herein may alsobe any organism belonging to the phylum Nematoda. In some instances, thenematode is considered a pest, e.g., an agricultural pest. For example,the nematode may be parasitic or cause health problems to plant or tofungi (for example species of the orders Aphelenchida, Meloidogyne,Tylenchida and others) or to humans and animals (for example species ofthe orders Trichinellida, Tylenchida, Rhabditina, and Spirurida).

Plant nematodes encompass plant parasitic nematodes and nematodes livingin the soil. Plant parasitic nematodes include, but are not limited to,ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorusspp.; semiparasites such as Tylenchulus spp.; migratory endoparasitessuch as Pratylenchus spp., Radopholus spp., and Scutellonema spp.;sedentary parasites such as Heterodera spp., Globodera spp., andMeloidogyne spp., and stem and leaf endoparasites such as Ditylenchusspp., Aphelenchoides spp., and Hirshmaniella spp. Especially harmfulroot parasitic soil nematodes are such as cystforming nematodes of thegenera Heterodera or Globodera, and/or root knot nematodes of the genusMeloidogyne. Harmful species of these genera are for example Meloidogyneincognita, Heterodera glycines (soybean cyst nematode), Globoderapallida and Globodera rostochiensis (potato cyst nematode), whichspecies are effectively controlled with the modulating agents describedherein. However, the use of the modulating agents described herein is inno way restricted to these genera or species, but also extends in thesame manner to other nematodes.

Plant nematodes include but are not limited to e.g. Aglenchus agricola,Anguina tritici, Aphelenchoides arachidis, Aphelenchoides fragaria andthe stem and leaf endoparasites Aphelenchoides spp. in general,Belonolaimus gracilis, Belonolaimus longicaudatus, Belonolaimus nortoni,Bursaphelenchus cocophilus, Bursaphelenchus eremus, Bursaphelenchusxylophilus, Bursaphelenchus mucronatus, and Bursaphelenchus spp. ingeneral, Cacopaurus pestis, Criconemella curvata, Criconemella onoensis,Criconemella ornata, Criconemella rusium, Criconemella xenoplax(=Mesocriconema xenoplax) and Criconemella spp. in general,Criconemoides femiae, Criconemoides onoense, Criconemoides ornatum andCriconemoides spp. in general, Ditylenchus destructor, Ditylenchusdipsaci, Ditylenchus myceliophagus and the stem and leaf endoparasitesDitylenchus spp. in general, Dolichodorus heterocephalus, Globoderapallida (=Heterodera pallida), Globodera rostochiensis (potato cystnematode), Globodera solanacearum, Globodera tabacum, Globodera virginiaand the sedentary, cyst forming parasites Globodera spp. in general,Helicotylenchus digonicus, Helicotylenchus dihystera, Helicotylenchuserythrine, Helicotylenchus multicinctus, Helicotylenchus nannus,Helicotylenchus pseudorobustus and Helicotylenchus spp. in general,Hemicriconemoides, Hemicycliophora arenaria, Hemicycliophora nudata,Hemicycliophora parvana, Heterodera avenae, Heterodera cruciferae,Heterodera glycines (soybean cyst nematode), Heterodera oryzae,Heterodera schachtii, Heterodera zeae and the sedentary, cyst formingparasites Heterodera spp. in general, Hirschmaniella gracilis,Hirschmaniella oryzae Hirschmaniella spinicaudata and the stem and leafendoparasites Hirschmaniella spp. in general, Hoplolaimus aegyptii,Hoplolaimus califomicus, Hoplolaimus columbus, Hoplolaimus galeatus,Hoplolaimus indicus, Hoplolaimus magnistylus, Hoplolaimus pararobustus,Longidorus africanus, Longidorus breviannulatus, Longidorus elongatus,Longidorus laevicapitatus, Longidorus vineacola and the ectoparasitesLongidorus spp. in general, Meloidogyne acronea, Meloidogyne africana,Meloidogyne arenaria, Meloidogyne arenaria thamesi, Meloidogyneartiella, Meloidogyne chitwoodi, Meloidogyne coffeicola, Meloidogyneethiopica, Meloidogyne exigua, Meloidogyne fallax, Meloidogynegraminicola, Meloidogyne graminis, Meloidogyne hapla, Meloidogyneincognita, Meloidogyne incognita acrita, Meloidogyne javanica,Meloidogyne kikuyensis, Meloidogyne minor, Meloidogyne naasi,Meloidogyne paranaensis, Meloidogyne thamesi and the sedentary parasitesMeloidogyne spp. in general, Meloinema spp., Nacobbus aberrans,Neotylenchus vigissi, Paraphelenchus pseudoparietinus, Paratrichodorusallius, Paratrichodorus lobatus, Paratrichodorus minor, Paratrichodorusnanus, Paratrichodorus porosus, Paratrichodorus teres andParatrichodorus spp. in general, Paratylenchus hamatus, Paratylenchusminutus, Paratylenchus projectus and Paratylenchus spp. in general,Pratylenchus agilis, Pratylenchus alleni, Pratylenchus andinus,Pratylenchus brachyurus, Pratylenchus cerealis, Pratylenchus coffeae,Pratylenchus crenatus, Pratylenchus delattrei, Pratylenchusgiibbicaudatus, Pratylenchus goodeyi, Pratylenchus hamatus, Pratylenchushexincisus, Pratylenchus loosi, Pratylenchus neglectus, Pratylenchuspenetrans, Pratylenchus pratensis, Pratylenchus scribneri, Pratylenchusteres, Pratylenchus thornei, Pratylenchus vulnus, Pratylenchus zeae andthe migratory endoparasites Pratylenchus spp. in general,Pseudohalenchus minutus, Psilenchus magnidens, Psilenchus tumidus,Punctodera chalcoensis, Quinisulcius acutus, Radopholus citrophilus,Radopholus similis, the migratory endoparasites Radopholus spp. ingeneral, Rotylenchulus borealis, Rotylenchulus parvus, Rotylenchulusreniformis and Rotylenchulus spp. in general, Rotylenchus laurentinus,Rotylenchus macrodoratus, Rotylenchus robustus, Rotylenchus uniformisand Rotylenchus spp. in general, Scutellonema brachyurum, Scutellonemabradys, Scutellonema clathricaudatum and the migratory endoparasitesScutellonema spp. in general, Subanguina radiciola, Tetylenchusnicotianae, Trichodorus cylindricus, Trichodorus minor, Trichodorusprimitivus, Trichodorus proximus, Trichodorus similis, Trichodorussparsus and the ectoparasites Trichodorus spp. in general,Tylenchorhynchus agri, Tylenchorhynchus brassicae, Tylenchorhynchusclarus, Tylenchorhynchus claytoni, Tylenchorhynchus digitatus,Tylenchorhynchus ebriensis, Tylenchorhynchus maximus, Tylenchorhynchusnudus, Tylenchorhynchus vulgaris and Tylenchorhynchus spp. in general,Tylenchulus semipenetrans and the semiparasites Tylenchulus spp. ingeneral, Xiphinema americanum, Xiphinema brevicolle, Xiphinemadimorphicaudatum, Xiphinema index and the ectoparasites Xiphinema spp.in general.

Other Examples of Nematode Hosts Include Species Belonging to the FamilyCriconematidae, Belonolaimidae, Hoploaimidae, Heteroderidae,Longidoridae, Pratylenchidae, Trichodoridae, or Anguinidae.

iv. Beneficial Hosts

In some instances, the host described herein is a beneficial insect,mollusk, or nematode (e.g., a pollinator, a natural competitor of apest, or a producer of useful substances for humans). The term“beneficial insect,” “beneficial mollusk,” or “beneficial nematode,” asused herein, refers to an insect, mollusk, or nematode that confers abenefit (e.g., economical and/or ecological) to humans, animals, anecosystem, and/or the environment. For example, the host may be aninvertebrate (e.g., insect, mollusk, or nematode) that is involved inthe production of a commercial product, including, but not limited to,invertebrates cultivated to produce food (e.g., honey from honey bees,e.g., Apis mellifera), materials (such as silk from Bombyx mori), and/orsubstances (e.g., lac from Laccifer lacca or pigments from Dactylopiuscoccus and Cynipidae). Additionally, the host may include invertebrates(e.g., insects, mollusks, or nematodes) that are used in agriculturalapplications, including invertebrates (e.g., insects, mollusks, ornematodes) that aid in the pollination of crops, spreading seeds, orpest control. Further, in some instances, the host may be aninvertebrate (e.g., insect, mollusk, or nematode) that is useful forwaste disposal and/or organic recycling (e.g., earthworms, termites, orDiptera larvae).

In some instances, the host produces a useable product (e.g., honey,silk, beeswax, or shellac). In some instances, the host is a bee.Exemplary bee genera include, but are not limited to Apis, Bombus,Trigona, and Osmia. In some instances, the bee is a honeybee (e.g., aninsect belonging to the genus Apis). In some instances, the honeybee isthe species Apis mellifera (the European or Western honey bee), Apiscerana (the Asiatic, Eastern, or Himalayan honey bee), Apis dorsata (the“giant” honey bee), Apis florea (the “red dwarf” honey bee), Apisandreniformis (the “black dwarf” honey bee), or Apis nigrocincta. Insome instances, the host is a silkworm. The silkworm may be a species inthe family Bombycidae or Saturniidae. In some instances, the silkworm isBombyx mori. In some instances, the host is a lac bug. The lac bug maybe a species in the family Kerriidae. In some instances, the lac bug isKerria lacca.

In some instances, the host aids in pollination of a plant (e.g., bees,beetles, wasps, flies, butterflies, or moths). In some examples, thehost aiding in pollination of a plant is beetle. In some instances, thebeetle is a species in the family Buprestidae, Cantharidae,Cerambycidae, Chrysomelidae, Cleridae, Coccinellidae, Elateridae,Melandryidae, Meloidae, Melyridae, Mordellidae, Nitidulidae,Oedemeridae, Scarabaeidae, or Staphyllinidae. In some instances, thehost aiding in pollination of a plant is a butterfly or moth (e.g.,Lepidoptera). In some instances, the butterfly or moth is a species inthe family Geometridae, Hesperiidae, Lycaenidae, Noctuidae, Nymphalidae,Papilionidae, Pieridae, or Sphingidae. In some instances, the hostaiding in pollination of a plant is a fly (e.g., Diptera). In someinstances, the fly is in the family Anthomyiidae, Bibionidae,Bombyliidae, Calliphoridae, Cecidomiidae, Certopogonidae, Chrionomidae,Conopidae, Culicidae, Dolichopodidae, Empididae, Ephydridae,Lonchopteridae, Muscidae, Mycetophilidae, Phoridae, Simuliidae,Stratiomyidae, or Syrphidae. In some instances, the host aiding inpollination is an ant (e.g., Formicidae), sawfly (e.g., Tenthredinidae),or wasp (e.g., Sphecidae or Vespidae). In some instances, the hostaiding in pollination of a plant is a bee. In some instances, the bee isin the family Andrenidae, Apidae, Colletidae, Halictidae, orMegachilidae.

In some instances, the host aids in pest control. In some instances, thehost aiding in pest control is a predatory nematode. In particularexamples, the nematode is a species of Heterorhabditis or Steinernema.In some instances, the host aiding in pest control is an insect. Forexample, the host aiding in pest control may be a species belonging tothe family Braconidae (e.g., parasitoid wasps), Carabidae (e.g., groundbeetles), Chrysopidae (e.g., green lacewings), Coccinellidae (e.g.,ladybugs), Hemerobiidae (e.g., brown lacewings), Ichneumonidae (e.g.,ichneumon wasps), Lampyridae (e.g., fireflies), Mantidae (e.g., prayingmantises), Myrmeleontidae (e.g., antilions), Odonata (e.g., dragonfliesand damselflies), or Syrphidae (e.g., hoverfly). In other instances, thehost aiding in pest control is an insect that competes with an insectthat is considered a pest (e.g., an agricultural pest). For example, theMediterranean fruit fly, Ceratitis capitata is a common pest of fruitsand vegetables worldwide. One way to control C. captitata is to releasethe sterilized male insect into the environment to compete with wildmales to mate the females. In these instances, the host may be asterilized male belonging to a species that is typically considered apest.

In some instances, the host aids in degradation of waste or organicmaterial. In some examples, the host aiding in degradation of waste ororganic material belongs to Coleoptera or Diptera. In some instances,the host belonging to Diptera is in the family Calliphoridae,Curtonotidae, Drosophilidae, Fanniidae, Heleomyzidae, Milichiidae,Muscidae, Phoridae, Psychodidae, Scatopsidae, Sepsidae, Sphaeroceridae,Stratiomyidae, Syrphidae, Tephritidae, or Ulidiidae. In some instances,the host belonging to Coleoptera is in the family Carabidae,Hydrophilidae, Phalacaridae, Ptiliidae, or Staphylinidae.

In some instances, the host may be an insect or an arachnid that may becultivated for a consumable product (e.g., food or feed). For example,the host may be a moth, butterfly, fly, cricket, spider, or beetle. Insome instances, the host is in the order Anoplura, Araneae, Blattodea,Coleoptera, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera,Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera,Isoptera, Lepidoptera, Mantodea, Mecoptera, Neuroptera, Odonata,Orthoptera, Phasmida, Plecoptera, Protura, Psocoptera, Siphonaptera,Siphunculata, Thysanura, Strepsiptera, Thysanoptera, Trichoptera, orZoraptera.

In some examples, the host is a black soldier fly (Hermetia illucens), acommon house fly, a lesser mealworm, a weaver ant, a silkworm (Bombyxmori), a grasshopper, a Chinese grasshopper (Acrida cinerea), a yellowmealworm (Clarias gariepinns), a moth (Anaphe infracta or Bombyx mori),Spodoptera littoralis, a house cricket, a termite, a palm weevil(Rhynchophorus ferruginens), a giant water bug (Lethocerus indicus), awater beetle, a termite (Macrotermes subhyalinus), a drugstore beetle(Stegobium paniceum), Imbrasia belina, Rhynchophorus phoenicis, Oryctesrhinoceros, Macrotermes bellicosus, Ruspolia differens, OryctesMonoceros, or Oecophylla smaragdina.

v. Decreasing Host Fitness

The methods and compositions provided herein may be used to decrease thefitness of any of the host invertebrates (e.g., insects, mollusks, ornematodes) described herein. The decrease in fitness arises fromalterations in host pathways that mediate interactions between the hostand microorganisms resident in the host, wherein the alterations are aconsequence of administration of a modulating agent and have detrimentaleffects on the host.

In some instances, the decrease in host fitness may manifest as adeterioration or decline in the physiology of the host (e.g., reducedhealth or survival) as a consequence of administration of a modulatingagent. In some instances, the fitness of an organism may be measured byone or more parameters, including, but not limited to, reproductiverate, fertility, lifespan, viability, mobility, fecundity, hostdevelopment, body weight, metabolic rate or activity, or survival incomparison to a host organism to which the modulating agent has not beenadministered. For example, the methods or compositions provided hereinmay be effective to decrease the overall health of the host or todecrease the overall survival of the host. In some instances, thedecreased survival of the host is about 2%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, or greater than 100% greater relative to areference level (e.g., a level found in a host that does not receive amodulating agent). In some instances, the methods and compositions areeffective to decrease host reproduction (e.g., reproductive rate,fertility) in comparison to a host organism to which the modulatingagent has not been administered. In some instances, the methods andcompositions are effective to decrease other physiological parameters,such as mobility, body weight, life span, fecundity, or metabolic rate,by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, orgreater than 100% relative to a reference level (e.g., a level found ina host that does not receive a modulating agent).

In some instances, the decrease in host fitness may manifest as adecrease in the production of one or more nutrients in the host (e.g.,vitamins, carbohydrates, amino acids, or polypeptides) in comparison toa host organism to which the modulating agent has not been administered.In some instances, the methods or compositions provided herein may beeffective to decrease the production of nutrients in the host (e.g.,vitamins, carbohydrates, amino acids, or polypeptides) by about 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%relative to a reference level (e.g., a level found in a host that doesnot receive a modulating agent). In some instances, the methods orcompositions provided herein may decrease nutrients in the host bydecreasing the production of nutrients by one or more microorganisms(e.g., endosymbiont) in the host in comparison to a host organism towhich the modulating agent has not been administered.

In some instances, the decrease in host fitness may manifest as anincrease in the host's sensitivity to a pesticidal agent (e.g., apesticide listed in Table 11) and/or a decrease in the host's resistanceto a pesticidal agent (e.g., a pesticide listed in Table 11) incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to increase the host's sensitivity to apesticidal agent (e.g., a pesticide listed in Table 11) by about 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%relative to a reference level (e.g., a level found in a host that doesnot receive a modulating agent). The pesticidal agent may be anypesticidal agent known in the art, including insecticidal agents. Insome instances, the methods or compositions provided herein may increasethe host's sensitivity to a pesticidal agent (e.g., a pesticide listedin Table 11) by decreasing the host's ability to metabolize or degradethe pesticidal agent into usable substrates in comparison to a hostorganism to which the modulating agent has not been administered.

In some instances, the decrease in host fitness may manifest as anincrease in the host's sensitivity to an allelochemical agent and/or adecrease in the host's resistance to an allelochemical agent incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to decrease the host's resistance to anallelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or greater than 100% relative to a reference level(e.g., a level found in a host that does not receive a modulatingagent). In some instances, the allelochemical agent is caffeine,soyacystatin, fenitrothion, monoterpenes, diterpene acids, or phenoliccompounds (e.g., tannins, flavonoids). In some instances, the methods orcompositions provided herein may increase the host's sensitivity to anallelochemical agent by decreasing the host's ability to metabolize ordegrade the allelochemical agent into usable substrates in comparison toa host organism to which the modulating agent has not been administered.

In some instances, the methods or compositions provided herein may beeffective to decease the host's resistance to parasites or pathogens(e.g., fungal, bacterial, or viral pathogens or parasites) in comparisonto a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to decrease the host's resistance to a pathogenor parasite (e.g., fungal, bacterial, or viral pathogens; or parasiticmites) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or greater than 100% relative to a reference level (e.g., a levelfound in a host that does not receive a modulating agent).

In some instances, the methods or compositions provided herein may beeffective to decrease the host's ability to carry or transmit a plantpathogen (e.g., plant virus (e.g., TYLCV) or a plant bacterium (e.g.,Agrobacterium spp)) in comparison to a host organism to which themodulating agent has not been administered. For example, the methods orcompositions provided herein may be effective to decrease the host'sability to carry or transmit a plant pathogen (e.g., a plant virus(e.g., TYLCV) or plant bacterium (e.g., Agrobacterium spp)) by about 2%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than100% relative to a reference level (e.g., a level found in a host thatdoes not receive a modulating agent).

In some instances, the decrease in host fitness may manifest as otherfitness disadvantages, such as a decreased tolerance to certainenvironmental factors (e.g., a high or low temperature tolerance), adecreased ability to survive in certain habitats, or a decreased abilityto sustain a certain diet in comparison to a host organism to which themodulating agent has not been administered. In some instances, themethods or compositions provided herein may be effective to decreasehost fitness in any plurality of ways described herein. Further, themodulating agent may decrease host fitness in any number of hostclasses, orders, families, genera, or species (e.g., 1 host species, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 200, 250, 500, or more host species). In some instances, themodulating agent acts on a single host class, order, family, genus, orspecies.

Host fitness may be evaluated using any standard methods in the art. Insome instances, host fitness may be evaluated by assessing an individualhost. Alternatively, host fitness may be evaluated by assessing a hostpopulation. For example, a decrease in host fitness may manifest as adecrease in successful competition against other insects, therebyleading to a decrease in the size of the host population.

vi. Increasing Host Fitness

The methods and compositions provided herein may be used to increase thefitness of any of the hosts described herein. The increase in fitnessarises from alterations in host pathways that mediate interactionsbetween the host and microorganisms resident in the host, wherein thealterations are a consequence of administration of a modulating agentand have beneficial effects on the host.

In some instances, the increase in host fitness may manifest as animprovement in the physiology of the host (e.g., improved health orsurvival) as a consequence of administration of a modulating agent. Insome instances, the fitness of an organism may be measured by one ormore parameters, including, but not limited to, reproductive rate,lifespan, mobility, fecundity, body weight, metabolic rate or activity,or survival in comparison to a host organism to which the modulatingagent has not been administered. For example, the methods orcompositions provided herein may be effective to improve the overallhealth of the host or to improve the overall survival of the host incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the improved survival of the host isabout 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, orgreater than 100% greater relative to a reference level (e.g., a levelfound in a host that does not receive a modulating agent). In someinstances, the methods and compositions are effective to increase hostreproduction (e.g., reproductive rate) in comparison to a host organismto which the modulating agent has not been administered. In someinstances, the methods and compositions are effective to increase otherphysiological parameters, such as mobility, body weight, life span,fecundity, or metabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a referencelevel (e.g., a level found in a host that does not receive a modulatingagent).

In some instances, the increase in host fitness may manifest as anincreased production of a product generated by said host in comparisonto a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to increase the production of a productgenerated by the host, as described herein (e.g., honey, beeswax,beebread, propolis, silk, or lac), by about 2%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to areference level (e.g., a level found in a host that does not receive amodulating agent).

In some instances, the increase in host fitness may manifest as anincrease in the frequency or efficacy of a desired activity carried outby the host (e.g., pollination, predation on pests, seed spreading, orbreakdown of waste or organic material) in comparison to a host organismto which the modulating agent has not been administered. In someinstances, the methods or compositions provided herein may be effectiveto increase the frequency or efficacy of a desired activity carried outby the host (e.g., pollination, predation on pests, seed spreading, orbreakdown of waste or organic material) by about 2%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to areference level (e.g., a level found in a host that does not receive amodulating agent).

In some instances, the increase in host fitness may manifest as anincrease in the production of one or more nutrients in the host (e.g.,vitamins, carbohydrates, amino acids, or polypeptides) in comparison toa host organism to which the modulating agent has not been administered.In some instances, the methods or compositions provided herein may beeffective to increase the production of nutrients in the host (e.g.,vitamins, carbohydrates, amino acids, or polypeptides) by about 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%relative to a reference level (e.g., a level found in a host that doesnot receive a modulating agent). In some instances, the methods orcompositions provided herein may increase nutrients in the host byincreasing the production of nutrients by one or more microorganisms(e.g., endosymbiont) in the host in comparison to a host organism towhich the modulating agent has not been administered.

In some instances, the increase in host fitness may manifest as adecrease in the host's sensitivity to a pesticidal agent (e.g., apesticide listed in Table 11) and/or an increase in the host'sresistance to a pesticidal agent (e.g., a pesticide listed in Table 11)in comparison to a host organism to which the modulating agent has notbeen administered. In some instances, the methods or compositionsprovided herein may be effective to decrease the host's sensitivity to apesticidal agent (e.g., a pesticide listed in Table 11) by about 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%relative to a reference level (e.g., a level found in a host that doesnot receive a modulating agent). The pesticidal agent may be anypesticidal agent known in the art, including insecticidal agents. Insome instances, the pesticidal agent is a neonicotinoid. In someinstances, the methods or compositions provided herein may decrease thehost's sensitivity to a pesticidal agent (e.g., a pesticide listed inTable 11) by increasing the host's ability to metabolize or degrade thepesticidal agent into usable substrates in comparison to a host organismto which the modulating agent has not been administered.

In some instances, the increase in host fitness may manifest as adecrease in the host's sensitivity to an allelochemical agent and/or anincrease in the host's resistance to an allelochemical agent incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to increase the host's resistance to anallelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or greater than 100% relative to a reference level(e.g., a level found in a host that does not receive a modulatingagent). In some instances, the allelochemical agent is caffeine,soyacystatin, fenitrothion, monoterpenes, diterpene acids, or phenoliccompounds (e.g., tannins, flavonoids). In some instances, the methods orcompositions provided herein may decrease the host's sensitivity to anallelochemical agent by increasing the host's ability to metabolize ordegrade the allelochemical agent into usable substrates in comparison toa host organism to which the modulating agent has not been administered.

In some instances, the methods or compositions provided herein may beeffective to increase the host's resistance to parasites or pathogens(e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g.,Varroa destructor mite in honeybees)) in comparison to a host organismto which the modulating agent has not been administered. In someinstances, the methods or compositions provided herein may be effectiveto increase the host's resistance to a pathogen or parasite (e.g.,fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroadestructor mite in honeybees)) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a referencelevel (e.g., a level found in a host that does not receive a modulatingagent).

In some instances, the increase in host fitness may manifest as otherfitness advantages, such as improved tolerance to certain environmentalfactors (e.g., a high or low temperature tolerance), improved ability tosurvive in certain habitats, or an improved ability to sustain a certaindiet (e.g., an improved ability to metabolize soy vs corn) in comparisonto a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to increase host fitness in any plurality ofways described herein. Further, the modulating agent may increase hostfitness in any number of host classes, orders, families, genera, orspecies (e.g., 1 host species, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 200, 250, 500, or more hostspecies). In some instances, the modulating agent acts on a single hostclass, order, family, genus, or species.

Host fitness may be evaluated using any standard methods in the art. Insome instances, host fitness may be evaluated by assessing an individualhost. Alternatively, host fitness may be evaluated by assessing a hostpopulation. For example, an increase in host fitness may manifest as anincrease in successful competition against other insects, therebyleading to an increase in the size of the host population.

vii. Hosts in Agriculture

The modulating agents described herein may be useful to promote thegrowth of plants. For example, by reducing the fitness of harmfulinvertebrates (e.g., insects, mollusks, or nematodes), the modulatingagents provided herein may be effective to promote the growth of plantsthat are typically harmed by a host. Alternatively, by increasing thefitness of beneficial invertebrates (e.g., insects, mollusks, ornematodes), the modulating agents provided herein may be effective topromote the growth of plants that benefit from said hosts. Themodulating agent may be delivered to the plant using any of theformulations and delivery methods described herein, in an amount and fora duration effective to modulate (e.g., increase or decrease) hostfitness and thereby benefit the plant, e.g., increase crop growth,increase crop yield, decrease pest infestation, and/or decrease damageto plants. This may or may not involve direct application of themodulating agent to the plant. For example, in instances where theprimary host habitat is different than the region of plant growth, themodulating agent may be applied to either the primary host habitat, theplants of interest, or a combination of both.

In some instances, the plant may be an agricultural food crop, such as acereal, grain, legume, fruit, or vegetable crop, or a non-food crop,e.g., grasses, flowering plants, cotton, hay, hemp. The compositionsdescribed herein may be delivered to the crop any time prior to or afterharvesting the cereal, grain, legume, fruit, vegetable, or other crop.Crop yield is a measurement often used for crop plants and is normallymeasured in metric tons per hectare (or kilograms per hectare). Cropyield can also refer to the actual seed generation from the plant. Insome instances, the modulating agent may be effective to increase cropyield (e.g., increase metric tons of cereal, grain, legume, fruit, orvegetable per hectare and/or increase seed generation) by about 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparisonto a reference level (e.g., a crop to which the modulating agent has notbeen administered).

In some instances, the plant (e.g., crop) may be at risk of developing apest infestation (e.g., by an insect, mollusk, or nematode) or may havealready developed a pest infestation. The methods and compositionsdescribed herein may be used to reduce or prevent pest infestation insuch crops by reducing the fitness of invertebrates (e.g., insect,mollusk, or nematode) that infest the plants. In some instances, themodulating agent may be effective to reduce crop infestation (e.g.,reduce the number of plants infested, reduce the pest population size,reduce damage to plants) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., acrop to which the modulating agent has not been administered). In otherinstances, the modulating agent may be effective to prevent or reducethe likelihood of crop infestation by about 2%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a referencelevel (e.g., a crop to which the modulating agent has not beenadministered).

Any suitable plant tissues may benefit from the compositions and methodsdescribed herein, including, but not limited to, somatic embryos,pollen, leaves, stems, calli, stolons, microtubers, or shoots. Themethods and compositions described herein may include treatment ofangiosperm or gymnosperm plants such as acacia, alfalfa, apple, apricot,artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet,birch, beech, blackberry, blueberry, broccoli, brussels sprouts,cabbage, canola, cantaloupe, carrot, cassaya, cauliflower, cedar, acereal, celery, chestnut, cherry, Chinese cabbage, citrus, clemintine,clover, coffee, corn, cotton, conifers, cowpea, cucumber, cypress,eggplant, elm, endive, eucalyptus, fava beans, fennel, forage crops,figs, fir, fruit and nut trees, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hemp, hickory, kale, kiwifruit, kohlrabi,larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize,mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, okra,onion, orange, an ornamental plant or flower or tree, papaya, palm,parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon,pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato,pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum,sallow, soybean, spinach, spruce, squash, strawberry, sugarbeet,sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco,tomato, trees, triticale, turf grasses, turnips, a vine, walnut,watercress, watermelon, wheat, yams, yew, or zucchini.

viii. Host in Feed/Food Production

Upon reaching a desired life stage, the host may be harvested and, ifdesired, processed for use in the manufacture of a consumable product.In some instances, the harvested invertebrate host (e.g., insect,mollusk, or nematode) may be distributed in a whole form (e.g., as thewhole, unprocessed host) as a consumable product. In some instances, thewhole harvested host is processed (e.g., ground up) and distributed as aconsumable product. Alternatively, one or more parts of the host (e.g.,one or more body parts or one or more substances) may be extracted fromthe host for use in the manufacture of a consumable product.

The consumable product may be any product safe for human or animalconsumption (e.g., ingestion). In some instances, the host may be usedin the manufacture of a feed for an animal. In some instances, theanimal is livestock or a farm animal (e.g., chicken, cow, horse, orpig). In some instances, the animal is a bird, reptile, amphibian,mammal, or fish. In some instances, the host may be used in themanufacture of a product that replaces the normal feed of an animal.Alternatively, the host may be used in the manufacture of a product thatsupplements the normal feed of an animal. The host may also be used inthe manufacture of a food, food additive, or food ingredient for humans.In some instances, the host is used in the manufacture of a nutritionalsupplement (e.g., protein supplement) for humans.

The host may be a wild or domesticated host. Additionally, the host maybe at any developmental stage at the time of delivering or applying thecompositions described herein. Further, the host may be at anydevelopmental stage at the time of harvesting the host for use in themanufacture of a consumable product. In some instances, the host is alarva, pupa, or adult insect at the time of harvesting, using,processing, or manufacturing. The delivery of the modulating agent andthe harvesting steps may occur at the same time or different times.

In some instances, a host species is selected based upon their naturalnutritional profile. In some instances, the modulating agent is used toimprove the nutritional profile of the insect, wherein the modulatingagent leads to an increased production of a nutrient in comparison to ahost organism to which the modulating agent has not been administered.Examples of nutrients include vitamins, carbohydrates, amino acids,polypeptides, or fatty acids. In some instances, the increasedproduction may arise from increased production of a nutrient by amicroorganism resident in the host. Alternatively, the increasedproduction may arise from increased production of a nutrient by the hostinsect itself, wherein the host has increased fitness following deliveryor administration of a modulating agent.

In some instances, in final processing, a first insect species iscombined with a second insect species whose nutritional profile providesa complementary benefit to the overall nutritional value of the food orfeed product. For example, a species containing a high protein profilecould be combined with a species containing a high omega 3/6 fatty acidprofile. In this manner, host protein meal may be custom blended to suitthe needs of humans or different species of animals.

ix. Host Insects in Disease Transmission

By decreasing the fitness of host insects that carry human and/or animalpathogens, the modulating agents provided herein may be effective toreduce the spread of vector-borne diseases. The modulating agent may bedelivered to the hosts using any of the formulations and deliverymethods described herein, in an amount and for a duration effective toreduce transmission of the disease, e.g., reduce vertical or horizontaltransmission between vectors and/or reduce transmission to humans and/oranimals. For example, the modulating agent described herein may reducevertical or horizontal transmission of a vector-borne pathogen by about2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more incomparison to a host organism to which the modulating agent has not beenadministered. As an another example, the modulating agent describedherein may reduce vectorial competence of an host vector by about 2%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more incomparison to a host organism to which the modulating agent has not beenadministered.

Non-limiting examples of diseases that may be controlled by thecompositions and methods provided herein include diseases caused byTogaviridae viruses (e.g., Chikungunya, Ross River fever, Mayaro,Onyon-nyong fever, Sindbis fever, Eastern equine enchephalomyeltis,Wesetern equine encephalomyelitis, Venezualan equine encephalomyelitis,or Barmah forest); diseases caused by Flavivirdae viruses (e.g., Denguefever, Yellow fever, Kyasanur Forest disease, Omsk haemorrhagic fever,Japaenese encephalitis, Murray Valley encephalitis, Rocio, St. Louisencephalitis, West Nile encephalitis, or Tick-borne encephalitis);diseases caused by Bunyaviridae viruses (e.g., Sandly fever, Rift Valleyfever, La Crosse encephalitis, California encephalitis, Crimean-Congohaemorrhagic fever, or Oropouche fever); disease caused by Rhabdoviridaeviruses (e.g., Vesicular stomatitis); disease caused by Orbiviridae(e.g., Bluetongue); diseases caused by bacteria (e.g., Plague,Tularaemia, Q fever, Rocky Mountain spotted fever, Murine typhus,Boutonneuse fever, Queensland tick typhus, Siberian tick typhus, Scrubtyphus, Relapsing fever, or Lyme disease); or diseases caused byprotozoa (e.g., Malaria, African trypanosomiasis, Nagana, Chagasdisease, Leishmaniasis, Piroplasmosis, Bancroftian filariasis, orBrugian filariasis).

II. Target Microorganisms

The microorganisms targeted by the modulating agent described herein mayinclude any microorganism resident in or on an invertebrate host (e.g.,insect, mollusk, or nematode), including, but not limited to, anybacteria and/or fungi described herein. Microorganisms resident in thehost may include, for example, symbiotic (e.g., endosymbioticmicroorganisms that provide beneficial nutrients or enzymes to thehost), commensal, pathogenic, or parasitic microorganisms. A symbioticmicroorganism (e.g., bacteria or fungi) may be an obligate symbiont ofthe host or a facultative symbiont of the host. Microorganisms residentin the host may be acquired by any mode of transmission, includingvertical, horizontal, or multiple origins of transmission.

i. Bacteria

Exemplary bacteria that may be targeted in accordance with the methodsand compositions provided herein, include, but are not limited to,Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp,Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp,Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidusspp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp,Xylella spp (e.g., Xylella fastidiosa), Erwinia spp, Agrobacterium spp,Bacillus spp, Commensalibacter spp. (e.g., Commensalibacter intestini),Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacteriumspp, Acetobacter spp (e.g., Acetobacter pomorum), Cyanobacteria spp,Salmonella spp, Rhodococcus spp, Pseudomonas spp (e.g., Psuedomonasfulva or Pseudomonas mandelii, Pseudomonas migulae), Pantoea spp. (e.g.,Pantoea vagans), Lactobacillus spp (e.g., Lactobacillus plantarum),Lysobacter spp., Herbaspirillum spp., Enterococcus spp, Gluconobacterspp. (e.g., Gluconobacter morbifer), Alcaligenes spp, Hamiltonella spp.,Klebsiella spp, Paenibacillus spp, Serratia spp. (e.g., Serratiamarcescens), Rahnella spp. (e.g., Rahnella aquatilis), Arthrobacter spp,Azotobacter spp., Corynebacterium spp, Brevibacterium spp, Regiella spp.(e.g., Regiella insecticola), Thermus spp, Pseudomonas spp, Clostridiumspp, Mortierella spp. (e.g., Mortierella elongata) and Escherichia spp.In some instances, the targeted bacteria are species in the generaXenorhabdus spp., Photorhabdus spp., or Wolbachia spp. In someinstances, the targeted bacteria are species in the orderStreptomycetales, Rhizobiales, Pseudomonadales, Xanthomondadales,Sphingobacteriales, Chlorofelxales, Rhodospirllales, Enterobacteriales,Sphingomonadales, Gemmatimonadales, Micrococcales, Caulobacterales,Cytophagales, Firmicutes, Micromonosporales, Burkholderiales,Rickettsiales, Flavobacteriales, Acidimicroiales, Rhodocyclales, orBdellovibrionales. In some instances, the targeted bacteria areArmatimonadetes, Firmicutes, TM7, Bacteroidetes, Proteobacteria, orActinobacteria. In some instances, the targeted bacteria are bacteria inthe genera Lactococcus spp., Aeromonas spp., Pseudomonas spp.,Enterobacter spp., Citrobacter spp., Sulfurospillium spp., Phaeosphaeriaspp., or Mycosphaerella spp. In some instances, the bacteria targeted bythe modulating agent may be ones that can be transmitted from the host(e.g., insect, mollusk, or nematode) to a plant, including, but notlimited to, bacterial plant pathogens (e.g., Agrobacterium spp.).Non-limiting examples of bacteria that may be targeted by the methodsand compositions provided herein are shown in Table 1. In someinstances, the 16S rRNA sequence of the bacteria targeted by themodulating agent has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,99%, 99.9%, or 100% identity with a sequence listed in Table 1.

TABLE 1 Examples of Target Bacteria and Host Insects Primaryendosymbiont Host Location 16S rRNA Gamma proteobacteria Carsonellaruddii Psyllids bacteriocytes TATCCAGCCACAGGTTCCC (Psylloidea)CTACAGCTACCTTGTTACGA CTTCACCCCAGTTACAAATC ATACCGTTGTAATAGTAAAATTACTTATGATACAATTTAC TTCCATGGTGTGACGGGCG GTGTGTACAAGGCTCGAGAACGTATTCACCGTAACATTC TGATTTACGATTACTAGCGA TTCCAACTTCATGAAATCGAGTTACAGATTTCAATCCGAA CTAAGAATATTTTTTAAGAT TAGCATTATGTTGCCATATAGCATATAACTTTTTGTAATA CTCATTGTAGCACGTGTGT AGCCCTACTTATAAGGGCCATGATGACTTGACGTCGTC CTCACCTTCCTCCAATTTAT CATTGGCAGTTTCTTATTAGTTCTAATATATTTTTAGTAAA ATAAGATAAGGGTTGCGCT CGTTATAGGACTTAACCCAACATTTCACAACACGAGCTG ACGACAGCCATGCAGCACC TGTCTCAAAGCTAAAAAAGCTTTATTATTTCTAATAAATTC TTTGGATGTCAAAAGTAGGT AAGATTTTTCGTGTTGTATCGAATTAAACCACATGCTCCA CCGCTTGTGCGAGCCCCCG TCAATTCATTTGAGTTTTAACCTTGCGGTCGTAATCCCC AGGCGGTCAACTTAACGCG TTAGCTTTTTCACTAAAAATATATAACTTTTTTTCATAAAA CAAAATTACAATTATAATATT TAATAAATAGTTGACATCGTTTACTGCATGGACTACCAG GGTATCTAATCCTGTTTGCT CCCCATGCTTTCGTGTATTAGTGTCAGTATTAAAATAGAA ATACGCCTTCGCCACTAGT ATTCTTTCAGATATCTAAGCATTTCACTGCTACTCCTGAA ATTCTAATTTCTTCTTTTATA CTCAAGTTTATAAGTATTAATTTCAATATTAAATTACTTTA ATAAATTTAAAAATTAATTTT TAAAAACAACCTGCACACCCTTTACGCCCAATAATTCCG ATTAACGCTTGCACCCCTC GTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGTGCT TCTTTTACAAATAACGTCAA AGATAATATTTTTTTATTATAAAATCTCTTCTTACTTTGTT GAAAGTGTTTTACAACCCTA AGGCCTTCTTCACACACGCGATATAGCTGGATCAAGCT TTCGCTCATTGTCCAATATC CCCCACTGCTGCCTTCCGTAAAAGTTTGGGCCGTGTCT CAGTCCCAATGTGGTTGTT CATCCTCTAAGATCAACTACGAATCATAGTCTTGTTAAGC TTTTACTTTAACAACTAACT AATTCGATATAAGCTCTTCTATTAGCGAACGACATTCTC GTTCTTTATCCATTAGGATA CATATTGAATTACTATACATTTCTATATACTTTTCTAATAC TAATAGGTAGATTCTTATAT ATTACTCACCCGTTCGCTGCTAATTATTTTTTTAATAATT CGCACAACTTGCATGTGTT AAGCTTATCGCTAGCGTTCAATCTGAGCTATGATCAAAC TCA (SEQ ID NO: 1) Portiera aleyrodidarumwhiteflyes bacteriocytes AAGAGTTTGATCATGGCTC BT-B (Aleyrodoidea)AGATTGAACGCTAGCGGCA GACATAACACATGCAAGTC GAGCGGCATCATACAGGTTGGCAAGCGGCGCACGGGT GAGTAATACATGTAAATATA CCTAAAAGTGGGGAATAACGTACGGAAACGTACGCTAA TACCGCATAATTATTACGAG ATAAAGCAGGGGCTTGATAAAAAAAATCAACCTTGCGCT TTTAGAAAATTACATGCCGG ATTAGCTAGTTGGTAGAGTAAAAGCCTACCAAGGTAACG ATCCGTAGCTGGTCTGAGA GGATGATCAGCCACACTGGGACTGAGAAAAGGCCCAGA CTCCTACGGGAGGCAGCAG TGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGT CATGCCGCGTGTGTGAAGA AGGCCTTTGGGTTGTAAAGCACTTTCAGCGAAGAAGAA AAGTTAGAAAATAAAAAGTT ATAACTATGACGGTACTCGCAGAAGAAGCACCGGCTAA CTCCGTGCCAGCAGCCGC GGTAAGACGGAGGGTGCAAGCGTTAATCAGAATTACTG GGCGTAAAGGGCATGTAGG TGGTTTGTTAAGCTTTATGTGAAAGCCCTATGCTTAACAT AGGAACGGAATAAAGAACT GACAAACTAGAGTGCAGAAGAGGAAGGTAGAATTCCCG GTGTAGCGGTGAAATGCGT AGATATCTGGAGGAATACCAGTTGCGAAGGCGACCTTC TGGGCTGACACTGACACTG AGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC CTGGTAGTCCACGCTGTAA ACGATATCAACTAGCCGTTGGATTCTTAAAGAATTTTGT GGCGTAGCTAACGCGATAA GTTGATCGCCTGGGGAGTACGGTCGCAAGGCTAAAACT CAAATGAATTGACGGGGGC CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCA ACGCGCAAAACCTTACCTA CTCTTGACATCCAAAGTACTTTCCAGAGATGGAAGGGTG CCTTAGGGAACTTTGAGAC AGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAAT GTTGGGTTAAGTCCCGTAA CGAGCGCAACCCTTGTCCTTAGTTGCCAACGCATAAGG CGGGAACTTTAAGGAGACT GCTGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAA GTCATCATGGCCCTTAAGA GTAGGGCAACACACGTGCTACAATGGCAAAAACAAAGG GTCGCAAAATGGTAACATG AAGCTAATCCCAAAAAAATTGTCTTAGTTCGGATTGGAG TCTGAAACTCGACTCCATAA AGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACG GTGAATACGTTCTCGGGCC TTGTACACACCGCCCGTCACACCATGGAAGTGAAATGC ACCAGAAGTGGCAAGTTTA ACCAAAAAACAGGAGAACAGTCACTACGGTGTGGTTCA TGACTGGGGTGAAGTCGTA ACAAGGTAGCTGTAGGGGAACCTGTGGCTGGATCACCT CCTTAA (SEQ ID NO: 2) Buchnera aphidicola Aphidsbacteriocytes AGAGTTTGATCATGGCTCA str. APS (Aphidoidea)GATTGAACGCTGGCGGCAA (Acyrthosiphon pisum) GCCTAACACATGCAAGTCGAGCGGCAGCGAGAAGAGA GCTTGCTCTCTTTGTCGGC AAGCGGCAAACGGGTGAGTAATATCTGGGGATCTACCC AAAAGAGGGGGATAACTAC TAGAAATGGTAGCTAATACCGCATAATGTTGAAAAACCAA AGTGGGGGACCTTTTGGCC TCATGCTTTTGGATGAACCCAGACGAGATTAGCTTGTTG GTAGAGTAATAGCCTACCA AGGCAACGATCTCTAGCTGGTCTGAGAGGATAACCAGC CACACTGGAACTGAGACAC GGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATT GCACAATGGGCGAAAGCCT GATGCAGCTATGCCGCGTGTATGAAGAAGGCCTTAGGG TTGTAAAGTACTTTCAGCGG GGAGGAAAAAAATAAAACTAATAATTTTATTTCGTGACG TTACCCGCAGAAGAAGCAC CGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGG GTGCAAGCGTTAATCAGAA TTACTGGGCGTAAAGAGCGCGTAGGTGGTTTTTTAAGTC AGGTGTGAAATCCCTAGGC TCAACCTAGGAACTGCATTTGAAACTGGAAAACTAGAGT TTCGTAGAGGGAGGTAGAA TTCTAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGG AATACCCGTGGCGAAAGCG GCCTCCTAAACGAAAACTGACACTGAGGCGCGAAAGCG TGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCATGCCGTAAACGATGTCGACTT GGAGGTTGTTTCCAAGAGA AGTGACTTCCGAAGCTAACGCATTAAGTCGACCGCCTG GGGAGTACGGCCGCAAGG CTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAAT TCGATGCAACGCGAAAAACCTTACCTGGTCTTGACATCC ACAGAATTCTTTAGAAATAA AGAAGTGCCTTCGGGAGCTGTGAGACAGGTGCTGCATG GCTGTCGTCAGCTCGTGTT GTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC TTATCCCCTGTTGCCAGCG GTTCGGCCGGGAACTCAGAGGAGACTGCCGGTTATAAA CCGGAGGAAGGTGGGGAC GACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACA CACGTGCTACAATGGTTTAT ACAAAGAGAAGCAAATCTGCAAAGACAAGCAAACCTCA TAAAGTAAATCGTAGTCCG GACTGGAGTCTGCAACTCGACTCCACGAAGTCGGAATC GCTAGTAATCGTGGATCAG AATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACACCATGGGA GTGGGTTGCAAAAGAAGCAGGTATCCTAACCCTTTAAAA GGAAGGCGCTTACCACTTT GTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACC GTAGGGGAACCTGCGGTTG GATCACCTCCTT (SEQ ID NO: 3)Buchnera aphidicola Aphids bacteriocytes AAACTGAAGAGTTTGATCAT str. Sg(Schizaphis (Aphidoidea) GGCTCAGATTGAACGCTGG graminum)CGGCAAGCCTAACACATGC AAGTCGAGCGGCAGCGAAA AGAAAGCTTGCTTTCTTGTCGGCGAGCGGCAAACGGGT GAGTAATATCTGGGGATCT GCCCAAAAGAGGGGGATAACTACTAGAAATGGTAGCTAA TACCGCATAAAGTTGAAAAA CCAAAGTGGGGGACCTTTTTTAAAGGCCTCATGCTTTTG GATGAACCCAGACGAGATT AGCTTGTTGGTAAGGTAAAAGCTTACCAAGGCAACGAT CTCTAGCTGGTCTGAGAGG ATAACCAGCCACACTGGAACTGAGACACGGTCCAGACT CCTACGGGAGGCAGCAGT GGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCT ATGCCGCGTGTATGAAGAA GGCCTTAGGGTTGTAAAGTACTTTCAGCGGGGAGGAAA AAATTAAAACTAATAATTTTA TTTTGTGACGTTACCCGCAGAAGAAGCACCGGCTAACT CCGTGCCAGCAGCCGCGG TAATACGGAGGGTGCGAGCGTTAATCAGAATTACTGGG CGTAAAGAGCACGTAGGTG GTTTTTTAAGTCAGATGTGAAATCCCTAGGCTTAACCTA GGAACTGCATTTGAAACTG AAATGCTAGAGTATCGTAGAGGGAGGTAGAATTCTAGG TGTAGCGGTGAAATGCGTA GATATCTGGAGGAATACCCGTGGCGAAAGCGGCCTCCT AAACGAATACTGACACTGA GGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC CTGGTAGTCCATGCCGTAA ACGATGTCGACTTGGAGGTTGTTTCCAAGAGAAGTGAC TTCCGAAGCTAACGCGTTA AGTCGACCGCCTGGGGAGTACGGCCGCAAGGCTAAAAC TCAAATGAATTGACGGGGG CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGC AACGCGAAAAACCTTACCT GGTCTTGACATCCACAGAATTTTTTAGAAATAAAAAAGT GCCTTCGGGAACTGTGAGA CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAA TGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTTATCCCCTGTTGCCAGCGGTTCGG CCGGGAACTCAGAGGAGAC TGCCGGTTATAAACCGGAGGAAGGTGGGGACGACGTC AAGTCATCATGGCCCTTAC GACCAGGGCTACACACGTGCTACAATGGTTTATACAAAG AGAAGCAAATCTGTAAAGA CAAGCAAACCTCATAAAGTAAATCGTAGTCCGGACTGGA GTCTGCAACTCGACTCCAC GAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCA CGGTGAATACGTTCCCGGG CCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTT GCAAAAGAAGCAGATTTCC TAACCACGAAAGTGGAAGGCGTCTACCACTTTGTGATTC ATGACTGGGGTGAAGTCGT AACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACC TCCTTA (SEQ ID NO: 4) Buchnera aphidicola Aphidsbacteriocytes ACTTAAAATTGAAGAGTTTG str. Bp (Baizongia (Aphidoidea)ATCATGGCTCAGATTGAAC pistaciae) GCTGGCGGCAAGCTTAACA CATGCAAGTCGAGCGGCATCGAAGAAAAGTTTACTTTTC TGGCGGCGAGCGGCAAAC GGGTGAGTAACATCTGGGGATCTACCTAAAAGAGGGGG ACAACCATTGGAAACGATG GCTAATACCGCATAATGTTTTTAAATAAACCAAAGTAGGG GACTAAAATTTTTAGCCTTA TGCTTTTAGATGAACCCAGACGAGATTAGCTTGATGGTA AGGTAATGGCTTACCAAGG CGACGATCTCTAGCTGGTCTGAGAGGATAACCAGCCAC ACTGGAACTGAGATACGGT CCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCA CAATGGGCTAAAGCCTGAT GCAGCTATGCCGCGTGTATGAAGAAGGCCTTAGGGTTG TAAAGTACTTTCAGCGGGG AGGAAAGAATTATGTCTAATATACATATTTTGTGACGTTA CCCGAAGAAGAAGCACCGG CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTG CGAGCGTTAATCAGAATTA CTGGGCGTAAAGAGCACGTAGGCGGTTTATTAAGTCAG ATGTGAAATCCCTAGGCTTA ACTTAGGAACTGCATTTGAAACTAATAGACTAGAGTCTCA TAGAGGGAGGTAGAATTCT AGGTGTAGCGGTGAAATGCGTAGATATCTAGAGGAATA CCCGTGGCGAAAGCGACCT CCTAAATGAAAACTGACGCTGAGGTGCGAAAGCGTGG GGAGCAAACAGGATTAGAT ACCCTGGTAGTCCATGCTGTAAACGATGTCGACTTGGA GGTTGTTTCCTAGAGAAGT GGCTTCCGAAGCTAACGCATTAAGTCGACCGCCTGGGG AGTACGGTCGCAAGGCTAA AACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTG GAGCATGTGGTTTAATTCG ATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCATA GAATTTTTTAGAGATAAAAG AGTGCCTTAGGGAACTATGAGACAGGTGCTGCATGGCT GTCGTCAGCTCGTGTTGTG AAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTA TCCTTTGTTGCCATCAGGTT ATGCTGGGAACTCAGAGGAGACTGCCGGTTATAAACCG GAGGAAGGTGGGGATGAC GTCAAGTCATCATGGCCCTTACGACCAGGGCTACACAC GTGCTACAATGGCATATAC AAAGAGATGCAACTCTGCGAAGATAAGCAAACCTCATAA AGTATGTCGTAGTCCGGAC TGGAGTCTGCAACTCGACTCCACGAAGTAGGAATCGCT AGTAATCGTGGATCAGAAT GCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGC CCGTCACACCATGGGAGTG GGTTGCAAAAGAAGCAGGTAGCTTAACCAGATTATTTTA TTGGAGGGCGCTTACCACT TTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAA CCGTAGGGGAACCTGCGGT TGGATCACCTCCTTA (SEQ ID NO: 5)Buchnera aphidicola Aphids bacteriocytes ATGAGATCATTAATATATAA BCc(Aphidoidea) AAATCATGTTCCAATTAAAA AATTAGGACAAAATTTTTTACAGAATAAAGAAATTATTAA TCAGATAATTAATTTAATAA ATATTAATAAAAATGATAATATTATTGAAATAGGATCAGG ATTAGGAGCGTTAACTTTTC CTATTTGTAGAATCATTAAAAAAATGATAGTATTAGAAAT TGATGAAGATCTTGTGTTTT TTTTAACTCAAAGTTTATTTATTAAAAAATTACAAATTATAA TTGCTGATATTATAAAATTT GATTTTTGTTGTTTTTTTTCTTTACAGAAATATAAAAAATA TAGGTTTATTGGTAATTTAC CATATAATATTGCTACTATATTTTTTTTAAAAACAATTAAA TTTCTTTATAATATAATTGAT ATGCATTTTATGTTTCAAAAAGAAGTAGCAAAGAGATTA TTAGCTACTCCTGGTACTAA AGAATATGGTAGATTAAGTATTATTGCACAATATTTTTATA AGATAGAAACTGTTATTAAT GTTAATAAATTTAATTTTTTTCCTACTCCTAAAGTAGATTC TACTTTTTTACGATTTACTC CTAAATATTTTAATAGTAAATATAAAATAGATAAACATTTT TCTGTTTTAGAATTAATTAC TAGATTTTCTTTTCAACATAGAAGAAAATTTTTAAATAAT AATTTAATATCTTTATTTTCT ACAAAAGAATTAATTTCTTTAGATATTGATCCATATTCAA GAGCAGAAAATGTTTCTTTA ATTCAATATTGTAAATTAATGAAATATTATTTGAAAAGAA AAATTTTATGTTTAGATTAA (SEQ ID NO: 6) Buchneraaphidicola Aphids bacteriocytes TTATCTTATTTCACATATAC (Cinara tujafilina)(Aphidoidea) GTAATATTGCGCTGCGTGC ACGAGGATTTTTTTGAATTTCAGATATATTTGGTTTAATA CGTTTAATAAAACGTATTTT TTTTTTTATTTTTCTTATTTGCAATTCAGTAATAGGAAGTT TTTTAGGTATATTTGGATAA TTACTGTAATTCTTAATAAAGTTTTTTACAATCCTATCTT CAATAGAATGAAAACTAATA ATAGCAATTTTTGATCCGGAATGTAATATGTTAATAATAA TTTTTAATATTTTATGTAATT CATTTATTTCTTGGTTAATATATATTCGAAAAGCTTGAAAT GTTCTCGTAGCTGGATGTTT AAATTTGTCATATTTTGGGATTGATTTTTTTATGATTTGAA CTAACTCTAACGTGCTTGTT ATGGTTTTTTTTTTTATTTGTAATATGATGGCTCGGGATA TTTTTTTTGCGTATTTTTCTT CGCCAAAATTTTTTATTACCTGTTCTATTGTTTTTTGGTTT GTTTTTTTTAACCATTGACT AACTGATATTCCAGATTTAGGGTTCATACGCATATCTAAA GGTCCATCATTCATAAATGA AAATCCTCGGATACTAGAATTTAACTGTATTGAAGAAATA CCTAAATCTAATAATATTCC ATCTATTTTATCTCTATTTTTTTCTTTTTTTAATATTTTTTC AATATTAGAAAATTTACCTA AAAATATTTTAAATCGCGAATCTTTTATTTTTTTTCCGATT TTTATAGATTGTGGGTCTTG ATCAATACTATATAACTTTCCATTAACCCCTAATTCTTGA AGAATTGCTTTTGAATGACC ACCACCTCCAAATGTACAATCAACATATGTACCGTCTTTT TTTATTTTTAAGTATTGTATG ATTTCTTTTGTTAAAACAGGTTTATGAATCAT (SEQ ID NO: 7) Buchnera aphidicola Aphids bacteriocytesATGAAAAGTATAAAAACTTT str. G002 (Myzus (Aphidoidea) TAAAAAACACTTTCCTGTGApersicae) AAAAATATGGACAAAATTTT CTTATTAATAAAGAGATCAT AAAAAATATTGTTAAAAAAATTAATCCAAATATAGAACAA ACATTAGTAGAAATCGGAC CAGGATTAGCTGCATTAACTGAGCCCATATCTCAGTTATT AAAAGAGTTAATAGTTATTG AAATAGACTGTAATCTATTATATTTTTTAAAAAAACAACC ATTTTATTCAAAATTAATAGT TTTTTGTCAAGATGCTTTAAACTTTAATTATACAAATTTAT TTTATAAAAAAAATAAATTAA TTCGTATTTTTGGTAATTTACCATATAATATCTCTACATC TTTAATTATTTTTTTATTTCA ACACATTAGAGTAATTCAAGATATGAATTTTATGCTTCAA AAAGAAGTTGCTGCAAGAT TAATTGCATTACCTGGAAATAAATATTACGGTCGTTTGAG CATTATATCTCAATATTATT GTGATATCAAAATTTTATTAAATGTTGCTCCTGAAGATTT TTGGCCTATTCCGAGAGTT CATTCTATATTTGTAAATTTAACACCTCATCATAATTCTCC TTATTTTGTTTATGATATTAA TATTTTAAGCCTTATTACAAATAAGGCTTTCCAAAATAGA AGAAAAATATTACGTCATAG TTTAAAAAATTTATTTTCTGAAACAACTTTATTAAATTTAG ATATTAATCCCAGATTAAGA GCTGAAAATATTTCTGTTTTTCAGTATTGTCAATTAGCTA ATTATTTGTATAAAAAAAATT ATACTAAAAAAAATTAA (SEQ ID NO:8) Buchnera aphidicola Aphids bacteriocytes ATTATAAAAAATTTTAAAAAA str.Ak (Acyrthosiphon (Aphidoidea) CATTTTCCTTTAAAAAGGTA kondoi)TGGACAAAATTTTCTTGTCA ATACAAAAACTATTCAAAAG ATAATTAATATAATTAATCCAAACACCAAACAAACATTAGT GGAAATTGGACCTGGATTA GCTGCATTAACAAAACCAATTTGTCAATTATTAGAAGAAT TAATTGTTATTGAAATAGAT CCTAATTTATTGTTTTTATTAAAAAAACGTTCATTTTATTC AAAATTAACAGTTTTTTATC AAGACGCTTTAAATTTCAATTATACAGATTTGTTTTATAA GAAAAATCAATTAATTCGTG TTTTTGGAAACTTGCCATATAATATTTCTACATCTTTAATT ATTTCTTTATTCAATCATATT AAAGTTATTCAAGATATGAATTTTATGTTACAGAAAGAGG TTGCTGAAAGATTAATTTCT ATTCCTGGAAATAAATCTTATGGCCGTTTAAGCATTATTT CTCAGTATTATTGTAAAATT AAAATATTATTAAATGTTGTACCTGAAGATTTTCGACCTA TACCGAAAGTGCATTCTGTT TTTATCAATTTAACTCCTCATACCAATTCTCCATATTTTG TTTATGATACAAATATCCTC AGTTCTATCACAAGAAATGCTTTTCAAAATAGAAGGAAAA TTTTGCGTCATAGTTTAAAA AATTTATTTTCTGAAAAAGAACTAATTCAATTAGAAATTA ATCCAAATTTACGAGCTGAA AATATTTCTATCTTTCAGTATTGTCAATTAGCTGATTATTT ATATAAAAAATTAAATAATCT TGTAAAAATCAATTAA (SEQ ID NO:9) Buchnera aphidicola Aphids bacteriocytes ATGATACTAAATAAATATAA str. Ua(Uroleucon (Aphidoidea) AAAATTTATTCCTTTAAAAA ambrosiae)GATACGGACAAAATTTTCTT GTAAATAGAGAAATAATCAA AAATATTATCAAAATAATTAATCCTAAAAAAACGCAAACAT TATTAGAAATTGGACCGGG TTTAGGTGCGTTAACAAAACCTATTTGTGAATTTTTAAAT GAACTTATCGTCATTGAAAT AGATCCTAATATATTATCTTTTTTAAAGAAATGTATATTTT TTGATAAATTAAAAATATATT GTCATAATGCTTTAGATTTTAATTATAAAAATATATTCTAT AAAAAAAGTCAATTAATTCG TATTTTTGGAAATTTACCATATAATATTTCTACATCTTTAA TAATATATTTATTTCGGAAT ATTGATATTATTCAAGATATGAATTTTATGTTACAACAAG AAGTGGCTAAAAGATTAGTT GCTATTCCTGGTGAAAAACTTTATGGTCGTTTAAGTATTA TATCTCAATATTATTGTAATA TTAAAATATTATTACATATTCGACCTGAAAATTTTCAACCT ATTCCTAAAGTTAATTCAAT GTTTGTAAATTTAACTCCGCATATTCATTCTCCTTATTTTG TTTATGATATTAATTTATTAA CTAGTATTACAAAACATGCTTTTCAACATAGAAGAAAAAT ATTGCGTCATAGTTTAAGAA ATTTTTTTTCTGAGCAAGATTTAATTCATTTAGAAATTAAT CCAAATTTAAGAGCTGAAAA TGTTTCTATTATTCAATATTGTCAATTGGCTAATAATTTAT ATAAAAAACATAAACAGTTT ATTAATAATTAA (SEQ ID NO: 10)Buchnera aphidicola Aphids bacteriocytes ATGAAAAAGCATATTCCTAT (Aphisglycines) (Aphidoidea) AAAAAAATTTAGTCAAAATT TTCTTGTAGATTTGAGTGTGATTAAAAAAATAATTAAATTT ATTAATCCGCAGTTAAATGA AATATTGGTTGAAATTGGACCGGGATTAGCTGCTATCAC TCGACCTATTTGTGATTTGA TAGATCATTTAATTGTGATTGAAATTGATAAAATTTTATT AGATAGATTAAAACAGTTCT CATTTTATTCAAAATTAACAGTATATCATCAAGATGCTTT AGCATTTGATTACATAAAGT TATTTAATAAAAAAAATAAATTAGTTCGAATTTTTGGTAAT TTACCATATCATGTTTCTAC GTCTTTAATATTGCATTTATTTAAAAGAATTAATATTATTAA AGATATGAATTTTATGCTAC AAAAAGAAGTTGCTGAACGTTTAATTGCAACTCCAGGTA GTAAATTATATGGTCGTTTA AGTATTATTTCTCAATATTATTGTAATATAAAAGTTTTATT GCATGTGTCTTCAAAATGTT TTAAACCAGTTCCTAAAGTAGAATCAATTTTTCTTAATTT GACACCTTATACTGATTATT TCCCTTATTTTACTTATAATGTAAACGTTCTTAGTTATAT TACAAATTTAGCTTTTCAAA AAAGAAGAAAAATATTACGTCATAGTTTAGGTAAAATATT TTCTGAAAAAGTTTTTATAA AATTAAATATTAATCCCAAATTAAGACCTGAGAATATTTC TATATTACAATATTGTCAGT TATCTAATTATATGATAGAAAATAATATTCATCAGGAACA TGTTTGTATTTAA (SEQ ID NO: 11) Annandia pinicola(Phylloxeroidea) bacteriocytes AGATTGAACGCTGGCGGCA TGCCTTACACATGCAAGTCGAACGGTAACAGGTCTTCG GACGCTGACGAGTGGCGAA CGGGTGAGTAATACATCGGAACGTGCCCAGTCGTGGGG GATAACTACTCGAAAGAGT AGCTAATACCGCATACGATCTGAGGATGAAAGCGGGG GACCTTCGGGCCTCGCGC GATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGG GATAAAAGCTTACCAAGCC GACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACA CTGGAACTGAGATACGGTC CAGACTCTTACGGGAGGCAGCAGTGGGGAATATTGCAC AATGGGCGCAAGCCTGATG CAGCTATGTCGCGTGTATGAAGAAGACCTTAGGGTTGT AAAGTACTTTCGATAGCATA AGAAGATAATGAGACTAATAATTTTATTGTCTGACGTTAG CTATAGAAGAAGCACCGGC TAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGGTG CTAGCGTTAATCGGAATTAC TGGGCGTAAAGAGCATGTAGGTGGTTTATTAAGTCAGAT GTGAAATCCCTGGACTTAAT CTAGGAACTGCATTTGAAACTAATAGGCTAGAGTTTCGT AGAGGGAGGTAGAATTCTA GGTGTAGCGGTGAAATGCATAGATATCTAGAGGAATATC AGTGGCGAAGGCGACCTTC TGGACGATAACTGACGCTAAAATGCGAAAGCATGGGTA GCAAACAGGATTAGATACC CTGGTAGTCCATGCTGTAAACGATGTCGACTAAGAGGT TGGAGGTATAACTTTTAATC TCTGTAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTA CGGTCGCAAGGCTAAAACT CAAATGAATTGACGGGGGCCTGCACAAGCGGTGGAGCA TGTGGTTTAATTCGATGCAA CGCGTAAAACCTTACCTGGTCTTGACATCCACAGAATTT TACAGAAATGTAGAAGTGC AATTTGAACTGTGAGACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTTGTGAAATGTT GGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTTG TTACCATAAGATTTAAGGAA CTCAAAGGAGACTGCCGGTGATAAACTGGAGGAAGGCG GGGACGACGTCAAGTCATC ATGGCCCTTATGACCAGGGCTACACACGTGCTACAATG GCATATACAAAGAGATGCA ATATTGCGAAATAAAGCCAATCTTATAAAATATGTCCTAG TTCGGACTGGAGTCTGCAA CTCGACTCCACGAAGTCGGAATCGCTAGTAATCGTGGA TCAGCATGCCACGGTGAAT ATGTTTCCAGGCCTTGTACACACCGCCCGTCACACCATG GAAGTGGATTGCAAAAGAA GTAAGAAAATTAACCTTCTTAACAAGGAAATAACTTACCA CTTTGTGACTCATAACTGG GGTGA (SEQ ID NO: 12) Moranellaendobia (Coccoidea) bacteriocytes TCTTTTTGGTAAGGAGGTGATCCAACCGCAGGTTCCCC TACGGTTACCTTGTTACGAC TTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTC CTAAAAGGTTAGGCTACCT ACTTCTTTTGCAACCCACTTCCATGGTGTGACGGGCGGT GTGTACAAGGCCCGGGAAC GTATTCACCGTGGCATTCTGATCCACGATTACTAGCGA TTCCTACTTCATGGAGTCGA GTTGCAGACTCCAATCCGGACTACGACGCACTTTATGA GGTCCGCTAACTCTCGCGA GCTTGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGT AGCCCTACTCGTAAGGGCC ATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTAT CACCGGCAGTCTCCTTTGA GTTCCCGACCGAATCGCTGGCAAAAAAGGATAAGGGTT GCGCTCGTTGCGGGACTTA ACCCAACATTTCACAACACGAGCTGACGACAGCCATGC AGCACCTGTCTCAGAGTTC CCGAAGGTACCAAAACATCTCTGCTAAGTTCTCTGGATG TCAAGAGTAGGTAAGGTTC TTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTT GTGCGGGCCCCCGTCAATT CATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCG GTCGATTTAACGCGTTAACT ACGAAAGCCACAGTTCAAGACCACAGCTTTCAAATCGA CATAGTTTACGGCGTGGAC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTC GTACCTGAGCGTCAGTATT CGTCCAGGGGGCCGCCTTCGCCACTGGTATTCCTCCA GATATCTACACATTTCACCG CTACACCTGGAATTCTACCCCCCTCTACGAGACTCTAG CCTATCAGTTTCAAATGCAG TTCCTAGGTTAAGCCCAGGGATTTCACATCTGACTTAAT AAACCGCCTACGTACTCTTT ACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTA TTACCGCGGCTGCTGGCAC GGAGTTAGCCGGTGCTTCTTCTGTAGGTAACGTCAATCA ATAACCGTATTAAGGATATT GCCTTCCTCCCTACTGAAAGTGCTTTACAACCCGAAGG CCTTCTTCACACACGCGGC ATGGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCC CACTGCTGCCTCCCGTAGG AGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCAT CCTCTCAGACCAGCTAGGG ATCGTCGCCTAGGTAAGCTATTACCTCACCTACTAGCTA ATCCCATCTGGGTTCATCT GAAGGTGTGAGGCCAAAAGGTCCCCCACTTTGGTCTTA CGACATTATGCGGTATTAG CTACCGTTTCCAGCAGTTATCCCCCTCCATCAGGCAGAT CCCCAGACTTTACTCACCC GTTCGCTGCTCGCCGGCAAAAAAGTAAACTTTTTTCCGT TGCCGCTCAACTTGCATGT GTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCATGATCA AACTCTTCAATTAAA (SEQ ID NO: 13) Ishikawaellacapsulata (Heteroptera) bacteriocytes AAATTGAAGAGTTTGATCAT MpkobeGGCTCAGATTGAACGCTAG CGGCAAGCTTAACACATGC AAGTCGAACGGTAACAGAAAAAAGCTTGCTTTTTTGCTG ACGAGTGGCGGACGGGTG AGTAATGTCTGGGGATCTACCTAATGGCGGGGGATAAC TACTGGAAACGGTAGCTAA TACCGCATAATGTTGTAAAACCAAAGTGGGGGACCTTAT GGCCTCACACCATTAGATG AACCTAGATGGGATTAGCTTGTAGGTGGGGTAAAGGCT CACCTAGGCAACGATCCCT AGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGA GATACGGTCCAGACTCCTA CGGGAGGCAGCAGTGGGGAATCTTGCACAATGGGCGC AAGCCTGATGCAGCTATGT CGCGTGTATGAAGAAGGCCTTAGGGTTGTAAAGTACTTT CATCGGGGAAGAAGGATAT GAGCCTAATATTCTCATATATTGACGTTACCTGCAGAAG AAGCACCGGCTAACTCCGT GCCAGCAGCCGCGGTAACACGGAGGGTGCGAGCGTTAA TCGGAATTACTGGGCGTAA AGAGCACGTAGGTGGTTTATTAAGTCATATGTGAAATCC CTGGGCTTAACCTAGGAAC TGCATGTGAAACTGATAAACTAGAGTTTCGTAGAGGGAG GTGGAATTCCAGGTGTAGC GGTGAAATGCGTAGATATCTGGAGGAATATCAGAGGCG AAGGCGACCTTCTGGACGA AAACTGACACTCAGGTGCGAAAGCGTGGGGAGCAAACA GGATTAGATACCCTGGTAG TCCACGCTGTAAACAATGTCGACTAAAAAACTGTGAGC TTGACTTGTGGTTTTTGTAG CTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCG CAAGGTTAAAACTCAAATGA ATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTT TAATTCGATGCAACGCGAA AAACCTTACCTGGTCTTGACATCCAGCGAATTATATAGAA ATATATAAGTGCCTTTCGGG GAACTCTGAGACGCTGCATGGCTGTCGTCAGCTCGTGT TGTGAAATGTTGGGTTAAGT CCCGCAACGAGCGCCCTTATCCTCTGTTGCCAGCGGCA TGGCCGGGAACTCAGAGGA GACTGCCAGTATTAAACTGGAGGAAGGTGGGGATGAC GTCAAGTCATCATGGCCCT TATGACCAGGGCTACACACGTGCTACAATGGTGTATAC AAAGAGAAGCAATCTCGCA AGAGTAAGCAAAACTCAAAAAGTACATCGTAGTTCGGA TTAGAGTCTGCAACTCGAC TCTATGAAGTAGGAATCGCTAGTAATCGTGGATCAGAAT GCCACGGTGAATACGTTCT CTGGCCTTGTACACACCGCCCGTCACACCATGGGAGTA AGTTGCAAAAGAAGTAGGT AGCTTAACCTTTATAGGAGGGCGCTTACCACTTTGTGA TTTATGACTGGGGTGAAGT CGTAACAAGGTAACTGTAGGGGAACCTGTGGTTGGATT ACCTCCTTA (SEQ ID NO: 14) Baumannia sharpshooterbacteriocytes TTCAATTGAAGAGTTTGATC cicadellinicola leafhoppersATGGCTCAGATTGAACGCT (Cicadellinae) GGCGGTAAGCTTAACACATGCAAGTCGAGCGGCATCG GAAAGTAAATTAATTACTTT GCCGGCAAGCGGCGAACGGGTGAGTAATATCTGGGGA TCTACCTTATGGAGAGGGA TAACTATTGGAAACGATAGCTAACACCGCATAATGTCGT CAGACCAAAATGGGGGACC TAATTTAGGCCTCATGCCATAAGATGAACCCAGATGAGA TTAGCTAGTAGGTGAGATA ATAGCTCACCTAGGCAACGATCTCTAGTTGGTCTGAGA GGATGACCAGCCACACTGG AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAG TGGGGAATCTTGCACAATG GGGGAAACCCTGATGCAGCTATACCGCGTGTGTGAAGA AGGCCTTCGGGTTGTAAAG CACTTTCAGCGGGGAAGAAAATGAAGTTACTAATAATAA TTGTCAATTGACGTTACCCG CAAAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGC GGTAAGACGGAGGGTGCAA GCGTTAATCGGAATTACTGGGCGTAAAGCGTATGTAGG CGGTTTATTTAGTCAGGTGT GAAAGCCCTAGGCTTAACCTAGGAATTGCATTTGAAACT GGTAAGCTAGAGTCTCGTA GAGGGGGGGAGAATTCCAGGTGTAGCGGTGAAATGCG TAGAGATCTGGAAGAATAC CAGTGGCGAAGGCGCCCCCCTGGACGAAAACTGACGC TCAAGTACGAAAGCGTGGG GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGT AAACGATGTCGATTTGAAG GTTGTAGCCTTGAGCTATAGCTTTCGAAGCTAACGCAT TAAATCGACCGCCTGGGGA GTACGACCGCAAGGTTAAAACTCAAATGAATTGACGGG GGCCCGCACAAGCGGTGG AGCATGTGGTTTAATTCGATACAACGCGAAAAACCTTAC CTACTCTTGACATCCAGAGT ATAAAGCAGAAAAGCTTTAGTGCCTTCGGGAACTCTGAG ACAGGTGCTGCATGGCTGT CGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTATC CTTTGTTGCCAACGATTAAGTCGGGAACTCAAAGGAGAC TGCCGGTGATAAACCGGAG GAAGGTGAGGATAACGTCAAGTCATCATGGCCCTTACG AGTAGGGCTACACACGTGC TACAATGGTGCATACAAAGAGAAGCAATCTCGTAAGAG TTAGCAAACCTCATAAAGTG CATCGTAGTCCGGATTAGAGTCTGCAACTCGACTCTAT GAAGTCGGAATCGCTAGTA ATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGG CCTTGTACACACCGCCCGT CACACCATGGGAGTGTATTGCAAAAGAAGTTAGTAGCT TAACTCATAATACGAGAGG GCGCTTACCACTTTGTGATTCATAACTGGGGTGAAGTCG TAACAAGGTAACCGTAGGG GAACCTGCGGTTGGATCACCTCCTTACACTAAA (SEQ ID NO: 15) Sodalis like Rhopalus wider tissueATTGAACGCTGGCGGCAGG sapporensis tropism CCTAACACATGCAAGTCGAGCGGCAGCGGGAAGAAGC TTGCTTCTTTGCCGGCGAG CGGCGGACGGGTGAGTAATGTCTGGGGATCTGCCCGAT GGAGGGGGATAACTACTGG AAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAA GTGGGGGACCTTCGGGCC TCACACCATCGGATGAACCCAGGTGGGATTAGCTAGTA GGTGGGGTAATGGCTCACC TAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAG TCACACTGGAACTGAGACA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT TGCACAATGGGGGAAACCC TGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGG GTTGTAAAGCACTTTCAGC GGGGAGGAAGGCGATGGCGTTAATAGCGCTATCGATTG ACGTTACCCGCAGAAGAAG CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGG AGGGTGCGAGCGTTAATCG GAATTACTGGGCGTAAAGCGTACGCAGGCGGTCTGTTA AGTCAGATGTGAAATCCCC GGGCTCAACCTGGGAACTGCATTTGAAACTGGCAGGCT AGAGTCTCGTAGAGGGGG GTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATC TGGAGGAATACCGGTGGCG AAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTAC GAAAGCGTGGGGAGCAAAC AGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATG TCGATTTGAAGGTTGTGGC CTTGAGCCGTGGCTTTCGGAGCTAACGTGTTAAATCGA CCGCCTGGGGAGTACGGC CGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGC ACAAGCGGTGGAGCATGTG GTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTT GACATCCAGAGAACTTGGC AGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGT GCTGCATGGCTGTCGTCAG CTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTATCCTTTATT GCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGG TGATAAACCGGAGGAAGGT GGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGG GCTACACACGTGCTACAAT GGCGCATACAAAGAGAAGCGATCTCGCGAGAGTCAGCG GACCTCATAAAGTGCGTCG TAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGT CGGAATCGCTAGTAATCGT GGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTG TACACACCGCCCGTCACAC CATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACC TTCGGGAGGGCGCTTACCA CTTTGTGATTCATGACTGG GGTG (SEQ IDNO: 16) Hartigia pinicola The pine bacteriocytes AGATTTAACGCTGGCGGCAbark adelgid GGCCTAACACATGCAAGTC GAGCGGTACCAGAAGAAGC TTGCTTCTTGCTGACGAGCGGCGGACGGGTGAGTAAT GTATGGGGATCTGCCCGAC AGAGGGGGATAACTATTGGAAACGGTAGCTAATACCGC ATAATCTCTGAGGAGCAAA GCAGGGGAACTTCGGTCCTTGCGCTATCGGATGAACCC ATATGGGATTAGCTAGTAG GTGAGGTAATGGCTCCCCTAGGCAACGATCCCTAGCTG GTCTGAGAGGATGATCAGC CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGA GGCAGCAGTGGGGAATATT GCACAATGGGCGAAAGCCTGATGCAGCCATGCCGCGTG TATGAAGAAGGCTTTAGGG TTGTAAAGTACTTTCAGTCGAGAGGAAAACATTGATGCT AATATCATCAATTATTGACG TTTCCGACAGAAGAAGCACCGGCTAACTCCGTGCCAGC AGCCGCGGTAATACGGAGG GTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCA CGCAGGCGGTTAATTAAGT TAGATGTGAAAGCCCCGGGCTTAACCCAGGAATAGCAT ATAAAACTGGTCAACTAGA GTATTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTG AAATGCGTAGAGATGTGGA GGAATACCAGTGGCGAAGGCGGCCCCCTGGACAAAAAC TGACGCTCAAATGCGAAAG CGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA TGCTGTAAACGATGTCGATT TGGAGGTTGTTCCCTTGAGGAGTAGCTTCCGTAGCTAA CGCGTTAAATCGACCGCCT GGGGGAGTACGACTGCAAGGTTAAAACTCAAATGAATT GACGGGGGCCCGCACAAG CGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAA ACCTTACCTACTCTTGACAT CCAGATAATTTAGCAGAAATGCTTTAGTACCTTCGGGAA ATCTGAGACAGGTGCTGCA TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAG TCCCGCAACGAGCGCAACC CTTATCCTTTGTTGCCAGCGATTAGGTCGGGAACTCAAA GGAGACTGCCGGTGATAAA CCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGC CCTTACGAGTAGGGCTACA CACGTGCTACAATGGCATATACAAAGGGAAGCAACCTC GCGAGAGCAAGCGAAACTC ATAAATTATGTCGTAGTTCAGATTGGAGTCTGCAACTCG ACTCCATGAAGTCGGAATC GCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGT TCCCGGGCCTTGTACACAC CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTA GGTAACTTAACCTTATGGAA AGCGCTTACCACTTTGTGATTCATAACTGGGGTG (SEQ ID NO: 17) Wigglesworthia tsetse fly bacteriocytesglossinidia (Diptera: Glossinidae) Beta proteobacteria Tremblayaphenacola Phenacoccus bacteriomes AGGTAATCCAGCCACACCT avenaeTCCAGTACGGCTACCTTGT (TPPAVE). TACGACTTCACCCCAGTCA CAACCCTTACCTTCGGAACTGCCCTCCTCACAACTCAA ACCACCAAACACTTTTAAAT CAGGTTGAGAGAGGTTAGGCCTGTTACTTCTGGCAAGA ATTATTTCCATGGTGTGACG GGCGGTGTGTACAAGACCCGAGAACATATTCACCGTGG CATGCTGATCCACGATTACT AGCAATTCCAACTTCATGCACTCGAGTTTCAGAGTACAAT CCGAACTGAGGCCGGCTTT GTGAGATTAGCTCCCTTTTGCAAGTTGGCAACTCTTTGG TCCGGCCATTGTATGATGT GTGAAGCCCCACCCATAAAGGCCATGAGGACTTGACGT CATCCCCACCTTCCTCCAA CTTATCGCTGGCAGTCTCTTTAAGGTAACTGACTAATCCA GTAGCAATTAAAGACAGGG GTTGCGCTCGTTACAGGACTTAACCCAACATCTCACGAC ACGAGCTGACGACAGCCAT GCAGCACCTGTGCACTAATTCTCTTTCAAGCACTCCCG CTTCTCAACAGGATCTTAGC CATATCAAAGGTAGGTAAGGTTTTTCGCGTTGCATCGAA TTAATCCACATCATCCACTG CTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAACCT TGCGGCCGTACTCCCCAGG CGGTCGACTTGTGCGTTAGCTGCACCACTGAAAAGGAA AACTGCCCAATGGTTAGTC AACATCGTTTAGGGCATGGACTACCAGGGTATCTAATC CTGTTTGCTCCCCATGCTTT AGTGTCTGAGCGTCAGTAACGAACCAGGAGGCTGCCTA CGCTTTCGGTATTCCTCCA CATCTCTACACATTTCACTGCTACATGCGGAATTCTACCT CCCCCTCTCGTACTCCAGC CTGCCAGTAACTGCCGCATTCTGAGGTTAAGCCTCAGC CTTTCACAGCAATCTTAACA GGCAGCCTGCACACCCTTTACGCCCAATAAATCTGATTA ACGCTCGCACCCTACGTAT TACCGCGGCTGCTGGCACGTAGTTTGCCGGTGCTTATTC TTTCGGTACAGTCACACCA CCAAATTGTTAGTTGGGTGGCTTTCTTTCCGAACAAAAG TGCTTTACAACCCAAAGGC CTTCTTCACACACGCGGCATTGCTGGATCAGGCTTCCG CCCATTGTCCAAGATTCCTC ACTGCTGCCTTCCTCAGAAGTCTGGGCCGTGTCTCAGT CCCAGTGTGGCTGGCCGTC CTCTCAGACCAGCTACCGATCATTGCCTTGGGAAGCCA TTACCTTTCCAACAAGCTAA TCAGACATCAGCCAATCTCAGAGCGCAAGGCAATTGGT CCCCTGCTTTCATTCTGCTT GGTAGAGAACTTTATGCGGTATTAATTAGGCTTTCACCT AGCTGTCCCCCACTCTGAG GCATGTTCTGATGCATTACTCACCCGTTTGCCACTTGCC ACCAAGCCTAAGCCCGTGT TGCCGTTCGACTTGCATGTGTAAGGCATGCCGCTAGCG TTCAATCTGAGCCAGGATC AAACTCT (SEQ ID NO: 18)Tremblaya princeps citrus bacteriomes AGAGTTTGATCCTGGCTCA mealybugGATTGAACGCTAGCGGCAT Planococcus citri GCATTACACATGCAAGTCGTACGGCAGCACGGGCTTAG GCCTGGTGGCGAGTGGCG AACGGGTGAGTAACGCCTCGGAACGTGCCTTGTAGTGG GGGATAGCCTGGCGAAAGC CAGATTAATACCGCATGAAGCCGCACAGCATGCGCGG TGAAAGTGGGGGATTCTAG CCTCACGCTACTGGATCGGCCGGGGTCTGATTAGCTAG TTGGCGGGGTAATGGCCCA CCAAGGCTTAGATCAGTAGCTGGTCTGAGAGGACGATC AGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAA TCTTGGACAATGGGCGCAA GCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTT CGGGTCGTAAAGCACTTTT GTTCGGGATGAAGGGGGGCGTGCAAACACCATGCCCT CTTGACGATACCGAAAGAA TAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGGTGCGAGCGTTA ATCGGAATCACTGGGCGTAAAGGGTGCGCGGGTGGTTT GCCAAGACCCCTGTAAAAT CCTACGGCCCAACCGTAGTGCTGCGGAGGTTACTGGTA AGCTTGAGTATGGCAGAGG GGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAT ATCTGGAGGAATACCGAAG GCGAAGGCAACCCCCTGGGCCATCACTGACACTGAGG CACGAAAGCGTGGGGAGC AAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAAC CATGTCGACTAGTTGTCGG GGGGAGCCCTTTTTCCTCGGTGACGAAGCTAACGCATG AAGTCGACCGCCTGGGGA GTACGACCGCAAGGTTAAAACTCAAAGGAATTGACGGG GACCCGCACAAGCGGTGG ATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTAC CTACCCTTGACATGGCGGA GATTCTGCCGAGAGGCGGAAGTGCTCGAAAGAGAATCC GTGCACAGGTGCTGCATGG CTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCATAACGAGCGCAACCCC CGTCTTTAGTTGCTACCACTGGGGCACTCTATAGAGACT GCCGGTGATAAACCGGAGG AAGGTGGGGACGACGTCAAGTCATCATGGCCTTTATGG GTAGGGCTTCACACGTCAT ACAATGGCTGGAGCAAAGGGTCGCCAACTCGAGAGAGG GAGCTAATCCCACAAACCC AGCCCCAGTTCGGATTGCACTCTGCAACTCGAGTGCAT GAAGTCGGAATCGCTAGTA ATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGG TCTTGTACACACCGCCCGT CACACCATGGGAGTAAGCCGCATCAGAAGCAGCCTCCC TAACCCTATGCTGGGAAGG AGGCTGCGAAGGTGGGGTCTATGACTGGGGTGAAGTC GTAACAAGGTAGCCGTACC GGAAGGTGCGGCTGGATTA CCT (SEQ IDNO: 19) Vidania bacteriomes Nasuia pestiferous bacteriomesAGTTTAATCCTGGCTCAGAT deltocephalinicola insect host, TTAACGCTTGCGACATGCCMacrosteles TAACACATGCAAGTTGAAC quadripunctulatus GTTGAAAATATTTCAAAGTA(Hemiptera: GCGTATAGGTGAGTATAAC Cicadellidae) ATTTAAACATACCTTAAAGTTCGGAATACCCCGATGAAA ATCGGTATAATACCGTATAA AAGTATTTAAGAATTAAAGCGGGGAAAACCTCGTGCTAT AAGATTGTTAAATGCCTGAT TAGTTTGTTGGTTTTTAAGGTAAAAGCTTACCAAGACTTT GATCAGTAGCTATTCTGTGA GGATGTATAGCCACATTGGGATTGAAATAATGCCCAAAC CTCTACGGAGGGCAGCAGT GGGGAATATTGGACAATGAGCGAAAGCTTGATCCAGCA ATGTCGCGTGTGCGATTAA GGGAAACTGTAAAGCACTTTTTTTTAAGAATAAGAAATTT TAATTAATAATTAAAATTTTT GAATGTATTAAAAGAATAAGTACCGACTAATCACGTGCC AGCAGTCGCGGTAATACGT GGGGTGCGAGCGTTAATCGGATTTATTGGGCGTAAAGT GTATTCAGGCTGCTTAAAAA GATTTATATTAAATATTTAAATTAAATTTAAAAAATGTATAA ATTACTATTAAGCTAGAGTT TAGTATAAGAAAAAAGAATTTTATGTGTAGCAGTGAAATG CGTTGATATATAAAGGAAC GCCGAAAGCGAAAGCATTTTTCTGTAATAGAACTGACGC TTATATACGAAAGCGTGGG TAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCT AAACTATGTCAATTAACTAT TAGAATTTTTTTTAGTGGTGTAGCTAACGCGTTAAATTGA CCGCCTGGGTATTACGATC GCAAGATTAAAACTCAAAGGAATTGACGGGGACCAGCA CAAGCGGTGGATGATGTGG ATTAATTCGATGATACGCGAAAAACCTTACCTGCCCTTGA CATGGTTAGAATTTTATTGA AAAATAAAAGTGCTTGGAAAAGAGCTAACACACAGGTGC TGCATGGCTGTCGTCAGCT CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCCTACTCTTAGTTGC TAATTAAAGAACTTTAAGAGAACAGCTAACAATAAGTTTA GAGGAAGGAGGGGATGAC TTCAAGTCCTCATGGCCCTTATGGGCAGGGCTTCACACG TCATACAATGGTTAATACAA AAAGTTGCAATATCGTAAGATTGAGCTAATCTTTAAAATT AATCTTAGTTCGGATTGTAC TCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGC GGTGAATAGTTTAACTGGTCTTGTACACACCGCCCGTC ACACCATGGAAATAAATCTT GTTTTAAATGAAGTAATATATTTTATCAAAACAGGTTTTG TAACCGGGGTGAAGTCGTA ACA (SEQ ID NO: 20) Zinderiainsecticola spittlebug bacteriocytes ATATAAATAAGAGTTTGATC CARIClastoptera CTGGCTCAGATTGAACGCT arizonana AGCGGTATGCTTTACACATGCAAGTCGAACGACAATAT TAAAGCTTGCTTTAATATAA AGTGGCGAACGGGTGAGTAATATATCAAAACGTACCTTA AAGTGGGGGATAACTAATT GAAAAATTAGATAATACCGCATATTAATCTTAGGATGAAA ATAGGAATAATATCTTATGC TTTTAGATCGGTTGATATCTGATTAGCTAGTTGGTAGGG TAAATGCTTACCAAGGCAAT GATCAGTAGCTGGTTTTAGCGAATGATCAGCCACACTG GAACTGAGACACGGTCCAG ACTTCTACGGAAGGCAGCAGTGGGGAATATTGGACAAT GGGAGAAATCCTGATCCAG CAATACCGCGTGAGTGATGAAGGCCTTAGGGTCGTAAA ACTCTTTTGTTAGGAAAGAA ATAATTTTAAATAATATTTAAAATTGATGACGGTACCTAAA GAATAAGCACCGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGC GTTAATCGGAATTATTGGG CGTAAAGAGTGCGTAGGCTGTTATATAAGATAGATGTGA AATACTTAAGCTTAACTTAA GAACTGCATTTATTACTGTTTAACTAGAGTTTATTAGAGA GAAGTGGAATTTTATGTGTA GCAGTGAAATGCGTAGATATATAAAGGAATATCGATGG CGAAGGCAGCTTCTTGGAA TAATACTGACGCTGAGGCACGAAAGCGTGGGGAGCAAA CAGGATTAGATACCCTGGT AGTCCACGCCCTAAACTATGTCTACTAGTTATTAAATTA AAAATAAAATTTAGTAACGT AGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGATC GCAAGATTAAAACTCAAAG GAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGG ATTAATTCGATGCAACACGA AAAACCTTACCTACTCTTGACATGTTTGGAATTTTAAAGA AATTTAAAAGTGCTTGAAAA AGAACCAAAACACAGGTGCTGCATGGCTGTCGTCAGCT CGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTTGTTATTATTTGC TAATAAAAAGAACTTTAATA AGACTGCCAATGACAAATTGGAGGAAGGTGGGGATGA CGTCAAGTCCTCATGGCCC TTATGAGTAGGGCTTCACACGTCATACAATGATATATAC AATGGGTAGCAAATTTGTG AAAATGAGCCAATCCTTAAAGTATATCTTAGTTCGGATTG TAGTCTGCAACTCGACTAC ATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGC CGCGGTGAATACGTTCTCG GGTCTTGTACACACCGCCCGTCACACCATGGAAGTGAT TTTTACCAGAAATTATTTGT TTAACCTTTATTGGAAAAAAATAATTAAGGTAGAATTCAT GACTGGGGTGAAGTCGTAA CAAGGTAGCAGTATCGGAAGGTGCGGCTGGATTACATT TTAAAT (SEQ ID NO: 21) Profftella armaturaDiaphorina citri, bacteriomes the Asian citrus psyllid Alphaproteobacteria Hodgkinia Cicada bacteriome AATGCTGGCGGCAGGCCTADiceroprocta ACACATGCAAGTCGAGCGG semicincta ACAACGTTCAAACGTTGTTAGCGGCGAACGGGTGAGTA ATACGTGAGAATCTACCCAT CCCAACGTGATAACATAGTCAACACCATGTCAATAACGT ATGATTCCTGCAACAGGTA AAGATTTTATCGGGGATGGATGAGCTCACGCTAGATTA GCTAGTTGGTGAGATAAAA GCCCACCAAGGCCAAGATCTATAGCTGGTCTGGAAGGA TGGACAGCCACATTGGGAC TGAGACAAGGCCCAACCCTCTAAGGAGGGCAGCAGTGA GGAATATTGGACAATGGGC GTAAGCCTGATCCAGCCATGCCGCATGAGTGATTGAAG GTCCAACGGACTGTAAAAC TCTTTTCTCCAGAGATCATAAATGATAGTATCTGGTGATA TAAGCTCCGGCCAACTTCG TGCCAGCAGCCGCGGTAATACGAGGGGAGCGAGTATTG TTCGGTTTTATTGGGCGTAA AGGGTGTCCAGGTTGCTAAGTAAGTTAACAACAAAATCT TGAGATTCAACCTCATAACG TTCGGTTAATACTACTAAGCTCGAGCTTGGATAGAGACA AACGGAATTCCGAGTGTAG AGGTGAAATTCGTTGATACTTGGAGGAACACCAGAGGC GAAGGCGGTTTGTCATACC AAGCTGACACTGAAGACACGAAAGCATGGGGAGCAAAC AGGATTAGATACCCTGGTA GTCCATGCCCTAAACGTTGAGTGCTAACAGTTCGATCA AGCCACATGCTATGATCCA GGATTGTACAGCTAACGCGTTAAGCACTCCGCCTGGGT ATTACGACCGCAAGGTTAA AACTCAAAGGAATTGACGGAGACCCGCACAAGCGGTG GAGCATGTGGTTTAATTCG AAGCTACACGAAGAACCTTACCAGCCCTTGACATACCA TGGCCAACCATCCTGGAAA CAGGATGTTGTTCAAGTTAAACCCTTGAAATGCCAGGAA CAGGTGCTGCATGGCTGTT GTCAGTTCGTGTCGTGAGATGTATGGTTAAGTCCCAAAA CGAACACAACCCTCACCCA TAGTTGCCATAAACACAATTGGGTTCTCTATGGGTACTG CTAACGTAAGTTAGAGGAA GGTGAGGACCACAACAAGTCATCATGGCCCTTATGGGC TGGGCCACACACATGCTAC AATGGTGGTTACAAAGAGCCGCAACGTTGTGAGACCGA GCAAATCTCCAAAGACCAT CTCAGTCCGGATTGTACTCTGCAACCCGAGTACATGAA GTAGGAATCGCTAGTAATC GTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTT GTACACGCCGCCCGTCACA CCATGGGAGCTTCGCTCCGATCGAAGTCAAGTTACCCTT GACCACATCTTGGCAAGTG ACCGA (SEQ ID NO: 22) Wolbachiasp. wPip Mosquito bacteriome AAATTTGAGAGTTTGATCCT CulexGGCTCAGAATGAACGCTGG quinquefasciatus CGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATT GTAGCTTGCTATGGTATAAC TTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCT AGTAGTACGGAATAATTGTT GGAAACGACAACTAATACCGTATACGCCCTACGGGGGA AAAATTTATTGCTATTAGAT GAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCC TACCAAGGTAATGATCTATA GCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAG ATACGGTCCAGACTCCTAC GGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAA AGCCTGATCCAGCCATGCC GCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTT AGTGAGGAAGATAATGACG GTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGC AGCCGCGGTAATACGGAGA GGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCG CGTAGGCTGGTTAATAAGT TAAAAGTGAAATCCCGAGGCTTAACCTTGGAATTGCTTT TAAAACTATTAATCTAGAGA TTGAAAGAGGATAGAGGAATTCCTGATGTAGAGGTAAAA TTCGTAAATATTAGGAGGAA CACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACG CTGAAGCGCGAAGGCGTG GGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCT GTAAACGATGAATGTTAAAT ATGGGGAGTTTACTTTCTGTATTACAGCTAACGCGTTAAA CATTCCGCCTGGGGACTAC GGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACC CGCACAAGCGGTGGAGCAT GTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTT CTTGACATGAAAATCATACC TATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTTACA CAAGTGTTGCATGGCTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTCATCC TTAGTTGCCATCAGGTAATGCTGAGTACTTTAAGGAAACT GCCAGTGATAAGCTGGAGG AAGGTGGGGATGATGTCAAGTCATCATGGCCTTTATGG AGTGGGCTACACACGTGCT ACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGCC TAAGCTAATCCCTAAAAGAC ATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGA AGTTGGAATCGCTAGTAAT CGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTC TTGTACACACTGCCCGTCA CGCCATGGGAATTGGTTTCACTCGAAGCTAATGGCCTA ACCGCAAGGAAGGAGTTAT TTAAAGTGGGATCAGTGACTGGGGTGAAGTCGTAACAA GGTAGCAGTAGGGGAATCT GCAGCTGGATTACCTCCTTA (SEQ ID NO:23) Bacteroidetes Uzinura diaspidicola armoured bacteriocytesAAAGGAGATATTCCAACCA scale insects CACCTTCCGGTACGGTTACCTTGTTACGACTTAGCCCTA GTCATCAAGTTTACCTTAGG CAGACCACTGAAGGATTACTGACTTCAGGTACCCCCGA CTCCCATGGCTTGACGGGC GGTGTGTACAAGGTTCGAGAACATATTCACCGCGCCATT GCTGATGCGCGATTACTAG CGATTCCTGCTTCATAGAGTCGAATTGCAGACTCCAATC CGAACTGAGACTGGTTTTA GAGATTAGCTCCTGATCACCCAGTGGCTGCCCTTTGTA ACCAGCCATTGTAGCACGT GTGTAGCCCAAGGCATAGAGGCCATGATGATTTGACAT CATCCCCACCTTCCTCACA GTTTACACCGGCAGTTTTGTTAGAGTCCCCGGCTTTACC CGATGGCAACTAACAATAG GGGTTGCGCTCGTTATAGGACTTAACCAAACACTTCACA GCACGAACTGAAGACAACC ATGCAGCACCTTGTAATACGTCGTATAGACTAAGCTGTT TCCAGCTTATTCGTAATACA TTTAAGCCTTGGTAAGGTTCCTCGCGTATCATCGAATTAA ACCACATGCTCCACCGCTT GTGCGAACCCCCGTCAATTCCTTTGAGTTTCAATCTTGC GACTGTACTTCCCAGGTGG ATCACTTATCGCTTTCGCTAAGCCACTGAATATCGTTTTT CCAATAGCTAGTGATCATC GTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTG CTCCCCACGCTTTCGTGCA CTGAGCGTCAGTAAAGATTTAGCAACCTGCCTTCGCTA TCGGTGTTCTGTATGATATC TATGCATTTCACCGCTACACCATACATTCCAGATGCTCCA ATCTTACTCAAGTTTACCAG TATCAATAGCAATTTTACAGTTAAGCTGTAAGCTTTCACT ACTGACTTAATAAACAGCCT ACACACCCTTTAAACCCAATAAATCCGAATAACGCTTGT GTCATCCGTATTGCCGCGG CTGCTGGCACGGAATTAGCCGACACTTATTCGTATAGTA CCTTCAATCTCCTATCACGT AAGATATTTTATTTCTATACAAAAGCAGTTTACAACCTAAA AGACCTTCATCCTGCACGC GACGTAGCTGGTTCAGAGTTTCCTCCATTGACCAATATT CCTCACTGCTGCCTCCCGT AGGAGTCTGGTCCGTGTCTCAGTACCAGTGTGGAGGTA CACCCTCTTAGGCCCCCTA CTGATCATAGTCTTGGTAGAGCCATTACCTCACCAACTAA CTAATCAAACGCAGGCTCA TCTTTTGCCACCTAAGTTTTAATAAAGGCTCCATGCAGA AACTTTATATTATGGGGGAT TAATCAGAATTTCTTCTGGCTATACCCCAGCAAAAGGTA GATTGCATACGTGTTACTCA CCCATTCGCCGGTCGCCGACAAATTAAAAATTTTTCGAT GCCCCTCGACTTGCATGTG TTAAGCTCGCCGCTAGCGTTAATTCTGAGCCAGGATCA AACTCTTCGTTGTAG (SEQ ID NO: 24) Sulcia muelleriBlue-Green bacteriocytes CTCAGGATAAACGCTAGCG SharpshooterGAGGGCTTAACACATGCAA and several GTCGAGGGGCAGCAAAAAT otherAATTATTTTTGGCGACCGG leafhopper CAAACGGGTGAGTAATACA speciesTACGTAACTTTCCTTATGCT GAGGAATAGCCTGAGGAAA CTTGGATTAATACCTCATAATACAATTTTTTAGAAAGAAA AATTGTTAAAGTTTTATTAT GGCATAAGATAGGCGTATGTCCAATTAGTTAGTTGGTAA GGTAATGGCTTACCAAGAC GATGATTGGTAGGGGGCCTGAGAGGGGCGTTCCCCCA CATTGGTACTGAGACACGG ACCAAACTTCTACGGAAGGCTGCAGTGAGGAATATTGG TCAATGGAGGAAACTCTGA ACCAGCCACTCCGCGTGCAGGATGAAAGAAAGCCTTAT TGGTTGTAAACTGCTTTTGT ATATGAATAAAAAATTCTAATTATAGAAATAATTGAAGGT AATATACGAATAAGTATCGA CTAACTCTGTGCCAGCAGTCGCGGTAAGACAGAGGATA CAAGCGTTATCCGGATTTAT TGGGTTTAAAGGGTGCGTAGGCGGTTTTTAAAGTCAGT AGTGAAATCTTAAAGCTTAA CTTTAAAAGTGCTATTGATACTGAAAAACTAGAGTAAGG TTGGAGTAACTGGAATGTG TGGTGTAGCGGTGAAATGCATAGATATCACACAGAACAC CGATAGCGAAAGCAAGTTA CTAACCCTATACTGACGCTGAGTCACGAAAGCATGGGG AGCAAACAGGATTAGATAC CCTGGTAGTCCATGCCGTAAACGATGATCACTAACTATT GGGTTTTATACGTTGTAATT CAGTGGTGAAGCGAAAGTGTTAAGTGATCCACCTGAGG AGTACGACCGCAAGGTTGA AACTCAAAGGAATTGACGGGGGCCCGCACAATCGGTG GAGCATGTGGTTTAATTCG ATGATACACGAGGAACCTTACCAAGACTTAAATGTACTA CGAATAAATTGGAAACAATT TAGTCAAGCGACGGAGTACAAGGTGCTGCATGGTTGTC GTCAGCTCGTGCCGTGAGG TGTAAGGTTAAGTCCTTTAAACGAGCGCAACCCTTATTA TTAGTTGCCATCGAGTAATG TCAGGGGACTCTAATAAGACTGCCGGCGCAAGCCGAG AGGAAGGTGGGGATGACGT CAAATCATCACGGCCCTTACGTCTTGGGCCACACACGT GCTACAATGATCGGTACAA AAGGGAGCGACTGGGTGACCAGGAGCAAATCCAGAAA GCCGATCTAAGTTCGGATT GGAGTCTGAAACTCGACTCCATGAAGCTGGAATCGCTA GTAATCGTGCATCAGCCAT GGCACGGTGAATATGTTCCCGGGCCTTGTACACACCGC CCGTCAAGCCATGGAAGTT GGAAGTACCTAAAGTTGGTTCGCTACCTAAGGTAAGTC TAATAACTGGGGCTAAGTC GTAACAAGGTA (SEQ ID NO: 25)Yeast like Symbiotaphrina Anobiid mycetome AGATTAAGCCATGCAAGTC buchnerivoucher beetles between the TAAGTATAAGNAATCTATAC JCM9740 Stegobiumforegut and NGTGAAACTGCGAATGGCT paniceum midgut CATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTAC ATGGATAACCGTGGTAATT CTAGAGCTAATACATGCTAAAAACCCCGACTTCGGAAGG GGTGTATTTATTAGATAAAA AACCAATGCCCTTCGGGGCTCCTTGGTGATTCATGATAA CTTAACGAATCGCATGGCC TTGCGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCA ACTTTCGATGGTAGGATAG TGGCCTACCATGGTTTTAACGGGTAACGGGGAATTAGGG TTCGATTCCGGAGAGGGAG CCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGG CGCGCAAATTACCCAATCC CGACACGGGGAGGTAGTGACAATAAATACTGATACAGG GCTCTTTTGGGTCTTGTAAT TGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATT GGAGGGCAAGTCTGGTGC CAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTA AAGTTGTTGCAGTTAAAAAG CTCGTAGTTGAACCTTGGGCCTGGCTGGCCGGTCCGC CTAACCGCGTGTACTGGTC CGGCCGGGCCTTTCCTTCTGGGGAGCCGCATGCCCTTC ACTGGGTGTGTCGGGGAAC CAGGACTTTTACTTTGAAAAAATTAGAGTGTTCAAAGCA GGCCTATGCTCGAATACAT TAGCATGGAATAATAGAATAGGACGTGCGGTTCTATTTT GTTGGTTTCTAGGACCGCC GTAATGATTAATAGGGATAGTCGGGGGCATCAGTATTCA ATTGTCAGAGGTGAAATTCT TGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCC AAGGATGTTTTCATTAATCA GTGAACGAAAGTTAGGGGATCGAAGACGATCAGATACC GTCGTAGTCTTAACCATAAA CTATGCCGACTAGGGATCGGGCGATGTTATTATTTTGAC TCGCTCGGCACCTTACGAG AAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCA AGGCTGAAACTTAAAGAAAT TGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTT AATTTGACTCAACACGGGG AAACTCACCAGGTCCAGACACATTAAGGATTGACAGATT GAGAGCTCTTTCTTGATTAT GTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAG TGATTTGTCTGCTTAATTGC GATAACGAACGAGACCTTAACCTGCTAAATAGCCCGGT CCGCTTTGGCGGGCCGCT GGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAG TTTGAGGCAATAACAGGTC TGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTA CACTGACAGAGCCAACGAG TAAATCACCTTGGCCGGAAGGTCTGGGTAATCTTGTTAA ACTCTGTCGTGCTGGGGAT AGAGCATTGCAATTATTGCTCTTCAACGAGGAATTCCTA GTAAGCGCAAGTCATCAGC TTGCGCTGATTACGTCCCTGCCCTTTGTACACACCGCC CGTCGCTACTACCGATTGA ATGGCTCAGTGAGGCCTTCGGACTGGCACAGGGACGTT GGCAACGACGACCCAGTGC CGGAAAGTTGGTCAAACTTGGTCATTTAGAGGAAGTAA AAGTCGTAACAAGGTTTCC GTAGGTGAACCTGCGGAAG GATCATTA(SEQ ID NO: 26) Symbiotaphrina kochii Anobiid mycetomeTACCTGGTTGATTCTGCCA voucher CBS 589.63 beetles GTAGTCATATGCTTGTCTCALasioderma AAGATTAAGCCATGCAAGT serricorne CTAAGTATAAGCAATCTATACGGTGAAACTGCGAATGGC TCATTAAATCAGTTATCGTT TATTTGATAGTACCTTACTACATGGATAACCGTGGTAAT TCTAGAGCTAATACATGCTA AAAACCTCGACTTCGGAAGGGGTGTATTTATTAGATAAA AAACCAATGCCCTTCGGGG CTCCTTGGTGATTCATGATAACTTAACGAATCGCATGGC CTTGCGCCGGCGATGGTTC ATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATA GTGGCCTACCATGGTTTCA ACGGGTAACGGGGAATTAGGGTTCGATTCCGGAGAGGG AGCCTGAGAAACGGCTACC ACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAAT CCCGACACGGGGAGGTAG TGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGT AATTGGAATGAGTACAATTT AAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGG TGCCAGCAGCCGCGGTAAT TCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAA AAAGCTCGTAGTTGAACCTT GGGCCTGGCTGGCCGGTCCGCCTAACCGCGTGTACTG GTCCGGCCGGGCCTTTCCT TCTGGGGAGCCGCATGCCCTTCACTGGGTGTGTCGGGG AACCAGGACTTTTACTTTGA AAAAATTAGAGTGTTCAAAGCAGGCCTATGCTCGAATAC ATTAGCATGGAATAATAGAA TAGGACGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGC CGTAATGATTAATAGGGATA GTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATTC TTGGATTTATTGAAGACTAA CTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATC AGTGAACGAAAGTTAGGGG ATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAA ACTATGCCGACTAGGGATC GGGCGATGTTATTATTTTGACTCGCTCGGCACCTTACGA GAAATCAAAGTCTTTGGGTT CTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGA AATTGACGGAAGGGCACCA CCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACG GGGAAACTCACCAGGTCCA GACACATTAAGGATTGACAGATTGAGAGCTCTTTCTTGA TTATGTGGGTGGTGGTGCA TGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAAT TGCGATAACGAACGAGACC TTAACCTGCTAAATAGCCCGGTCCGCTTTGGCGGGCC GCTGGCTTCTTAGAGGGAC TATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACAG GTCTGTGATGCCCTTAGAT GTTCTGGGCCGCACGCGCGCTACACTGACAGAGCCAA CGAGTACATCACCTTGGCC GGAAGGTCTGGGTAATCTTGTTAAACTCTGTCGTGCTG GGGATAGAGCATTGCAATT ATTGCTCTTCAACGAGGAATTCCTAGTAAGCGCAAGTCA TCAGCTTGCGCTGATTACG TCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCG ATTGAATGGCTCAGTGAGG CCTTCGGACTGGCACAGGGACGTTGGCAACGACGACCC AGTGCCGGAAAGTTCGTCA AACTTGGTCATTTAGAGGAAGNNNAAGTCGTAACAAGGT TTCCGTAGGTGAACCTGCG GAAGGATCATTA (SEQ ID NO: 27)Primary extracelullar symbiont Host location 16 rRNAfenitrothion-degrading bacteria Burkholderia sp. SFA1 Riptortus GutAGTTTGATCCTGGCTCAGA pedestris TTGAACGCTGGCGGCATGC CTTACACATGCAAGTCGAACGGCAGCACGGGGGCAAC CCTGGTGGCGAGTGGCGA ACGGGTGAGTAATACATCGGAACGTGTCCTGTAGTGGG GGATAGCCCGGCGAAAGC CGGATTAATACCGCATACGACCTAAGGGAGAAAGCGGG GGATCTTCGGACCTCGCGC TATAGGGGCGGCCGATGGCAGATTAGCTAGTTGGTGG GGTAAAGGCCTACCAAGGC GACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACA CTGGGACTGAGACACGGCC CAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGAC AATGGGGGCAACCCTGATC CAGCAATGCCGCGTGTGTGAAGAAGGCTTCGGGTTGTA AAGCACTTTTGTCCGGAAA GAAAACTTCGTCCCTAATATGGATGGAGGATGACGGTAC CGGAAGAATAAGCACCGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGC GAGCGTTAATCGGAATTAC TGGGCGTAAAGCGTGCGCAGGCGGTCTGTTAAGACCGA TGTGAAATCCCCGGGCTTA ACCTGGGAACTGCATTGGTGACTGGCAGGCTTTGAGTG TGGCAGAGGGGGGTAGAAT TCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGA ATACCGATGGCGAAGGCAG CCCCCTGGGCCAACTACTGACGCTCATGCACGAAAGCG TGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTA GTTGTTGGGGATTCATTTCC TTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGG GAGTACGGTCGCAAGATTA AAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGT GGATGATGTGGATTAATTC GATGCAACGCGAAAAACCTTACCTACCCTTGACATGGT CGGAACCCTGCTGAAAGGT GGGGGTGCTCGAAAGAGAACCGGCGCACAGGTGCTGC ATGGCTGTCGTCAGCTCGT GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA CCCTTGTCCTTAGTTGCTAC GCAAGAGCACTCTAAGGAGACTGCCGGTGACAAACCGG AGGAAGGTGGGGATGACGT CAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGT CATACAATGGTCGGAACAG AGGGTTGCCAAGCCGCGAGGTGGAGCCAATCCCAGAA AACCGATCGTAGTCCGGAT CGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCT AGTAATCGCGGATCAGCAT GCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGC CCGTCACACCATGGGAGTG GGTTTCACCAGAAGTAGGTAGCCTAACCGCAAGGAGGG CGCTTACCACGGTGGGATT CATGACTGGGGTGAAGTCG TAACAAGGTAGC(SEQ ID NO: 28) Burkholderia sp. KM-A Riptortus Gut GCAACCCTGGTGGCGAGTGpedestris GCGAACGGGTGAGTAATAC ATCGGAACGTGTCCTGTAG TGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCA TACGATCTACGGAAGAAAG CGGGGGATCCTTCGGGACCTCGCGCTATAGGGGCGG CCGATGGCAGATTAGCTAG TTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAG CTGGTCTGAGAGGACGACC AGCCACACTGGGACTGAGACACGGCCCAGACTCCTACG GGAGGCAGCAGTGGGGAA TTTTGGACAATGGGGGCAACCCTGATCCAGCAATGCCG CGTGTGTGAAGAAGGCCTT CGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAACGTCT TGGTTAATACCTGAGGCGG ATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGT GCCAGCAGCCGCGGTAATA CGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAA AGCGTGCGCAGGCGGTCT GTTAAGACCGATGTGAAATCCCCGGGCTTAACCTGGGA ACTGCATTGGTGACTGGCA GGCTTTGAGTGTGGCAGAGGGGGGTAGAATTCCACGTG TAGCAGTGAAATGCGTAGA GATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTG GGCCAACACTGACGCTCAT GCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCC TGGTAGTCCACGCCCTAAA CGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAA CGTAGCTAACGCGTGAAGT TGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAA AGGAATTGACGGGGACCCG CACAAGCGGTGGATGATGTGGATTAATTCGATGCAACG CGAAAAACCTTACCTACCCT TGACATGGTCGGAAGTCTGCTGAGAGGTGGACGTGCTC GAAAGAGAACCGGCGCACA GGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCAC TCTAAGGAGACTGCCGGTG ACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCA TGGCCCTTATGGGTAGGGC TTCACACGTCATACAATGGTCGGAACAGAGGGTTGCCAA GCCGCGAGGTGGAGCCAA TCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCA ACTCGACTGCGTGAAGCTG GAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAA TACGTTCCCGGGTCTTGTA CACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAG AAGTAGGTAGCCTAACCGC AAGGAGGGCGCTTACCACGGTGGGATTCATGACTGGGG TGAAGT (SEQ ID NO: 29) Burkholderia sp. KM-GRiptortus Gut GCAACCCTGGTGGCGAGTG pedestris GCGAACGGGTGAGTAATACATCGGAACGTGTCCTGTAG TGGGGGATAGCCCGGCGA AAGCCGGATTAATACCGCATACGACCTAAGGGAGAAAG CGGGGGATCTTCGGACCTC GCGCTATAGGGGCGGCCGATGGCAGATTAGCTAGTTG GTGGGGTAAAGGCCTACCA AGGCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGC CACACTGGGACTGAGACAC GGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTT GGACAATGGGGGCAACCCT GATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGG TTGTAAAGCACTTTTGTCCG GAAAGAAAACTTCGAGGTTAATACCCTTGGAGGATGAC GGTACCGGAAGAATAAGCA CCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG GGTGCGAGCGTTAATCGGA ATTACTGGGCGTAAAGCGTGCGCAGGCGGTCTGTTAAG ACCGATGTGAAATCCCCGG GCTTAACCTGGGAACTGCATTGGTGACTGGCAGGCTTT GAGTGTGGCAGAGGGGGG TAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGT GGAGGAATACCGATGGCGA AGGCAGCCCCCTGGGCCAACACTGACGCTCATGCACG AAAGCGTGGGGAGCAAACA GGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGT CAACTAGTTGTTGGGGATT CATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCG CCTGGGGAGTACGGTCGCA AGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAA GCGGTGGATGATGTGGATT AATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACA TGGTCGGAAGTCTGCTGAG AGGTGGACGTGCTCGAAAGAGAACCGGCGCACAGGTG CTGCATGGCTGTCGTCAGC TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC GCAACCCTTGTCCTTAGTT GCTACGCAAGAGCACTCTAAGGAGACTGCCGGTGACAA ACCGGAGGAAGGTGGGGA TGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTC ACACGTCATACAATGGTCG GAACAGAGGGTTGCCAAGCCGCGAGGTGGAGCCAATCC CAGAAAACCGATCGTAGTC CGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAA TCGCTAGTAATCGCGGATC AGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACAC ACCGCCCGTCACACCATGG GAGTGGGTTTCACCAGAAGTAGGTAGCCTAACCTGCAA AGGAGGGCGCTTACCACG (SEQ ID NO: 30) NematodesXiphinematobacter sp. Xiphinema ovaries, GCAAGTCGAACGGAGTGGA americanumdeveloping ACCTGCAGTAATGCAGATT eggs, and gut CGATTCAGTGGCGTACGGG liningTGCGTAACACGTGAGTGAT CTACCGGTAAGTGGGGGAT AACCCGCCGAAAGGCGAATTAATACCGCATGTGGCTAG GGATGCCTTCATCCTGTAG CTAAAGTCGATTTTGACGCTTTCTGATGAGCTCGCGGCC TATCAGCTTGTTGGTGGAG GTAATGGCCCACCAAGGCAATGACGGGTAGCTGGTCTG AGAGGACGATCAGCCACAC TGGAACTGAGACACGGTCCAGACACCTACGGGTGGCAG CAGTCGAGAATTTTTCACAA TGGGGGAAACCCTGATGAAGCAACGCCGCGTGGAGGA TGAAGGGCTTCGCGCTCGT AAACTCCTGTCAAGCGGGAACAAGAAAGTGATAGTACC GCTAGAGGAAGAGACGGCT AACTCTGTGCCAGCAGCCGCGGTAATACAGAGGTCTCG AGCGTTGTTCGGATTTATTG GGCGTAAAGGGTGCGTAGGCGGTGTGGCAAGTCAAGT GTGAA (SEQ ID NO: 31) Radopholus similis Wolbachiasp. wOo Onchocerca somatic ATGACACACATACCGGTTTT ochengi hypodermalACTAAAAGAAATGCTATCGC cords that run AACTTTCACCACAAAATGGT along theAGTGTATATGTGGATGCCA length of the CATTTGGAGCTGGAGGATA worms and inTAGTAAAGCAATATTGGAGT the germinal CAGCTGATTGCAGAGTGTA zones of theTGCAATCGACAGAGATGAA female gonad ACGGTTATTAAATTTTATAATAGTTTGAATACCAAGTACC ACGGTAAAATAAAACTATTT ATTGAAAAGTTTAGCAATATTCAAACTATACTAAACAGTA GTAATCTCAAACACTTTACA GAACCTTCCGTCATTGTTTCAGCTGGAATTCAGAAAAAA AATGCAAGGTCAAGCACCG AGATGATACAAAGTAATACCGTAGATGGAGTTGTGTTCG ATATAGGAGTATCGTCTATG CAGCTTGATGAAGAAAATAGAGGATTTTCATTTTTACAT AACAGTCCGCTTGATATGC GCATGGATACCTCTTCTCACATTAACGCTTCAATATTTG TTAATGCCTTACGCGAAGA AGAAATTGCAAACACTATATATAGCTATGGAGGTGAACG TTATTCTCGCAAAATTGCAA GAGCAATAGTGAACGTACGTAAGAAAAAAACTATCGACA CTACATTTGAGCTTGCAGA CATTGTACGTTCCGTGGTATCTCGCGGAAAAAGCAAGAT TGATCCTGCAACTAGGACA TTTCAAGCAATCAGAATATGGGTAAACGATGAGCTTAGA GAGCTTGAAAAGGGTATTA AAGCTGCATCCAAAATCTTAAATAGGAATGGCAAGCTGA TTGTCATTACTTTTCATTCC TTGGAAGATCGTATAGTCAAGACCTTTTTTAAAGGCTTAT GTGAGCCAAAATTCACCAA CTGTAGAACGTTTTCTCTTCTGAATAAAAAAGTAATCAAG GCAAGCGCAGAAGAAATAA GTGCAAATCCACGTGCGCGTTCAGCAAAACTAAGAGCTA TACAAAGGTTATTATGA (SEQ ID NO: 32) Snodgrassellaalvi Honeybee Ileum GAGAGTTTGATCCTGGCTC (Apis AGATTGAACGCTGGCGGCAmellifera) TGCCTTACACATGCAAGTC and Bombus GAACGGCAGCACGGAGAG spp.CTTGCTCTCTGGTGGCGAG TGGCGAACGGGTGAGTAAT GCATCGGAACGTACCGAGTAATGGGGGATAACTGTCCG AAAGGATGGCTAATACCGC ATACGCCCTGAGGGGGAAAGCGGGGGATCGAAAGACCT CGCGTTATTTGAGCGGCCG ATGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCA AGGCGACGATCCATAGCGG GTCTGAGAGGATGATCCGCCACATTGGGACTGAGACAC GGCCCAAACTCCTACGGGA GGCAGCAGTGGGGAATTTTGGACAATGGGGGGAACCCT GATCCAGCCATGCCGCGTG TCTGAAGAAGGCCTTCGGGTTGTAAAGGACTTTTGTTAG GGAAGAAAAGCCGGGTGTT AATACCATCTGGTGCTGACGGTACCTAAAGAATAAGCA CCGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGA ATTACTGGGCGTAAAGCGA GCGCAGACGGTTAATTAAGTCAGATGTGAAATCCCCGA GCTCAACTTGGGACGTGCA TTTGAAACTGGTTAACTAGAGTGTGTCAGAGGGAGGTAG AATTCCACGTGTAGCAGTG AAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGG CAGCCTCCTGGGATAACAC TGACGTTCATGCTCGAAAGCGTGGGTAGCAAACAGGAT TAGATACCCTGGTAGTCCA CGCCCTAAACGATGACAATTAGCTGTTGGGACACTAGA TGTCTTAGTAGCGAAGCTA ACGCGTGAAATTGTCCGCCTGGGGAGTACGGTCGCAAG ATTAAAACTCAAAGGAATTG ACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAA TTCGATGCAACGCGAAGAA CCTTACCTGGTCTTGACATGTACGGAATCTCTTAGAGA TAGGAGAGTGCCTTCGGGA ACCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGT GTCGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCAT CATTAAGTTGGGCACTCTAA TGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT GACGTCAAGTCCTCATGGC CCTTATGACCAGGGCTTCACACGTCATACAATGGTCGG TACAGAGGGTAGCGAAGCC GCGAGGTGAAGCCAATCTCAGAAAGCCGATCGTAGTCC GGATTGCACTCTGCAACTC GAGTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCA GCATACTGCGGTGAATACG TTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGG AGTGGGGGATACCAGAATT GGGTAGACTAACCGCAAGGAGGTCGCTTAACACGGTAT GCTTCATGACTGGGGTGAA GTCGTAACAAGGTAGCCGT AG (SEQ IDNO: 33) Gilliamella apicola honeybee Ileum TTAAATTGAAGAGTTTGATC (ApisATGGCTCAGATTGAACGCT mellifera) GGCGGCAGGCTTAACACAT and BombusGCAAGTCGAACGGTAACAT spp. GAGTGCTTGCACTTGATGA CGAGTGGCGGACGGGTGAGTAAAGTATGGGGATCTGC CGAATGGAGGGGGACAACA GTTGGAAACGACTGCTAATACCGCATAAAGTTGAGAGA CCAAAGCATGGGACCTTCG GGCCATGCGCCATTTGATGAACCCATATGGGATTAGCT AGTTGGTAGGGTAATGGCT TACCAAGGCGACGATCTCTAGCTGGTCTGAGAGGATGA CCAGCCACACTGGAACTGA GACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGG AATATTGCACAATGGGGGA AACCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCC TTCGGGTTGTAAAGTACTTT CGGTGATGAGGAAGGTGGTGTATCTAATAGGTGCATCAA TTGACGTTAATTACAGAAGA AGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATAC GGAGGGTGCGAGCGTTAAT CGGAATGACTGGGCGTAAAGGGCATGTAGGCGGATAAT TAAGTTAGGTGTGAAAGCC CTGGGCTCAACCTAGGAATTGCACTTAAAACTGGTTAAC TAGAGTATTGTAGAGGAAG GTAGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATG TGGAGGAATACCGGTGGCG AAGGCGGCCTTCTGGACAGATACTGACGCTGAGATGCG AAAGCGTGGGGAGCAAACA GGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGT CGATTTGGAGTTTGTTGCCT AGAGTGATGGGCTCCGAAGCTAACGCGATAAATCGACC GCCTGGGGAGTACGGCCG CAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACA AGCGGTGGAGCATGTGGTT TAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGA CATCCACAGAATCTTGCAG AGATGCGGGAGTGCCTTCGGGAACTGTGAGACAGGTGC TGCATGGCTGTCGTCAGCT CGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCTTATCCTTTGTTGC CATCGGTTAGGCCGGGAACTCAAAGGAGACTGCCGTTG ATAAAGCGGAGGAAGGTGG GGACGACGTCAAGTCATCATGGCCCTTACGACCAGGGC TACACACGTGCTACAATGG CGTATACAAAGGGAGGCGACCTCGCGAGAGCAAGCGG ACCTCATAAAGTACGTCTAA GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCG GAATCGCTAGTAATCGTGA ATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTA CACACCGCCCGTCACACCA TGGGAGTGGGTTGCACCAGAAGTAGATAGCTTAACCTTC GGGAGGGCGTTTACCACG GTGTGGTCCATGACTGGGGTGAAGTCGTAACAAGGTAA CCGTAGGGGAACCTGCGGT TGGATCACCTCCTTAC (SEQ ID NO: 34)Bartonella apis honeybee Gut AAGCCAAAATCAAATTTTCA (ApisACTTGAGAGTTTGATCCTG mellifera) GCTCAGAACGAACGCTGGC GGCAGGCTTAACACATGCAAGTCGAACGCACTTTTCGG AGTGAGTGGCAGACGGGT GAGTAACGCGTGGGAATCTACCTATTTCTACGGAATAAC GCAGAGAAATTTGTGCTAAT ACCGTATACGTCCTTCGGGAGAAAGATTTATCGGAGATA GATGAGCCCGCGTTGGATT AGCTAGTTGGTGAGGTAATGGCCCACCAAGGCGACGAT CCATAGCTGGTCTGAGAGG ATGACCAGCCACATTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGT GGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCC ATGCCGCGTGAGTGATGAA GGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAA TGACGGTAACCGGAGAAGA AGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATAC GAAGGGGGCTAGCGTTGTT CGGATTTACTGGGCGTAAAGCGCACGTAGGCGGATATT TAAGTCAGGGGTGAAATCC CGGGGCTCAACCCCGGAACTGCCTTTGATACTGGATAT CTTGAGTATGGAAGAGGTA AGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATT CGGAGGAACACCAGTGGC GAAGGCGGCTTACTGGTCCATTACTGACGCTGAGGTGC GAAAGCGTGGGGAGCAAAC AGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATG AATGTTAGCCGTTGGACAG TTTACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCC GCCTGGGGAGTACGGTCG CAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACA AGCGGTGGAGCATGTGGTT TAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGA CATCCCGATCGCGGATGGT GGAGACACCGTCTTTCAGTTCGGCTGGATCGGTGACAG GTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCGCCCTTA GTTGCCATCATTTAGTTGGGCACTCTAAGGGGACTGCC GGTGATAAGCCGAGAGGAA GGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGC TGGGCTACACACGTGCTAC AATGGTGGTGACAGTGGGCAGCGAGACCGCGAGGTCG AGCTAATCTCCAAAAGCCAT CTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAA GTTGGAATCGCTAGTAATC GTGGATCAGCATGCCACGGTGAATACGTTCCCGGGCCT TGTACACACCGCCCGTCAC ACCATGGGAGTTGGTTTTACCCGAAGGTGCTGTGCTAA CCGCAAGGAGGCAGGCAA CCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAA GGTAGCCGTAGGGGAACCT GCGGCTGGATCACCTCCTTTCTAAGGAAGATGAAGAATT GGAA (SEQ ID NO: 35) Parasaccharibacter honeybeeGut CTACCATGCAAGTCGCACG apium (Apis AAACCTTTCGGGGTTAGTG mellifera)GCGGACGGGTGAGTAACG CGTTAGGAACCTATCTGGA GGTGGGGGATAACATCGGGAAACTGGTGCTAATACCG CATGATGCCTGAGGGCCAA AGGAGAGATCCGCCATTGGAGGGGCCTGCGTTCGATTA GCTAGTTGGTTGGGTAAAG GCTGACCAAGGCGATGATCGATAGCTGGTTTGAGAGGA TGATCAGCCACACTGGGAC TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTG GGGAATATTGGACAATGGG GGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAG GTCTTCGGATTGTAAAGCA CTTTCACTAGGGAAGATGATGACGGTACCTAGAGAAGA AGCCCCGGCTAACTTCGTG CCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCT CGGAATGACTGGGCGTAAA GGGCGCGTAGGCTGTTTGTACAGTCAGATGTGAAATCC CCGGGCTTAACCTGGGAAC TGCATTTGATACGTGCAGACTAGAGTCCGAGAGAGGGT TGTGGAATTCCCAGTGTAG AGGTGAAATTCGTAGATATTGGGAAGAACACCGGTTGCG AAGGCGGCAACCTGGCTNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNN NNNNGAGCTAACGCGTTAAGCACACCGCCTGGGGAGTA CGGCCGCAAGGTTGAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC ATGTGGTTTAATTCGAAGCA ACGCGCAGAACCTTACCAGGGCTTGCATGGGGAGGCT GTATTCAGAGATGGATATTT CTTCGGACCTCCCGCACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTA GTTGCCATCACGTCTGGGT GGGCACTCTAGAGAGACTGCCGGTGACAAGCCGGAGG AAGGTGGGGATGACGTCAA GTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCT ACAATGGCGGTGACAGAGG GATGCTACATGGTGACATGGTGCTGATCTCAAAAAACC GTCTCAGTTCGGATTGTACT CTGCAACTCGAGTGCATGAAGGTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC ACACCATGGGAGTTGGTTT GACCTTAAGCCGGTGAGCGAACCGCAAGGAACGCAGCC GACCACCGGTTCGGGTTCA GCGACTGGGGGA (SEQ ID NO: 36)Lactobacillus kunkeei honeybee Gut TTCCTTAGAAAGGAGGTGA (ApisTCCAGCCGCAGGTTCTCCT mellifera) ACGGCTACCTTGTTACGAC TTCACCCTAATCATCTGTCCCACCTTAGACGACTAGCTC CTAAAAGGTTACCCCATCG TCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGT GTGTACAAGGCCCGGGAAC GTATTCACCGTGGCATGCTGATCCACGATTACTAGTGAT TCCAACTTCATGCAGGCGA GTTGCAGCCTGCAATCCGAACTGAGAATGGCTTTAAGA GATTAGCTTGACCTCGCGG TTTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTG TAGCCCAGCTCATAAGGGG CATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTT ATCACCGGCAGTCTCACTA GAGTGCCCAACTAAATGCTGGCAACTAATAATAAGGGT TGCGCTCGTTGCGGGACTT AACCCAACATCTCACGACACGAGCTGACGACAACCATG CACCACCTGTCATTCTGTC CCCGAAGGGAACGCCCAATCTCTTGGGTTGGCAGAAGA TGTCAAGAGCTGGTAAGGT TCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCAC TTGTGCGGGCCCCCGTCAA TTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGC GGAATACTTAATGCGTTAG CTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAG TATTCATCGTTTACGGCATG GACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCT TTCGAGCCTCAGCGTCAGT AACAGACCAGAAAGCCGCCTTCGCCACTGGTGTTCTTC CATATATCTACGCATTTCAC CGCTACACATGGAGTTCCACTTTCCTCTTCTGTACTCAA GTTTTGTAGTTTCCACTGCA CTTCCTCAGTTGAGCTGAGGGCTTTCACAGCAGACTTA CAAAACCGCCTGCGCTCGC TTTACGCCCAATAAATCCGGACAACGCTTGCCACCTAC GTATTACCGCGGCTGCTGG CACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAA AGTGTTAACAGTTACTCTAA CACTTGTTCTTCTTTAACAACAGAGTTTTACGATCCGAA AACCTTCATCACTCACGCG GCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATT CCCTACTGCTGCCTCCCGT AGGAGTCTGGGCCGTGTCTCAGTCCCAATGTGGCCGAT TACCCTCTCAGGTCGGCTA CGTATCATCGTCTTGGTGGGCTTTTATCTCACCAACTAA CTAATACGGCGCGGGTCCA TCCCAAAGTGATAGCAAAGCCATCTTTCAAGTTGGAACC ATGCGGTTCCAACTAATTAT GCGGTATTAGCACTTGTTTCCAAATGTTATCCCCCGCTTC GGGGCAGGTTACCCACGTG TTACTCACCAGTTCGCCACTCGCTCCGAATCCAAAAATC ATTTATGCAAGCATAAAATC AATTTGGGAGAACTCGTTCGACTTGCATGTATTAGGCA CGCCGCCAGCGTTCGTCCT GAGCCAGGATCAAACTCTC ATCTTAA (SEQID NO: 37) Lactobacillus Firm-4 honeybee Gut ACGAACGCTGGCGGCGTG (ApisCCTAATACATGCAAGTCGA mellifera) GCGCGGGAAGTCAGGGAA GCCTTCGGGTGGAACTGGTGGAACGAGCGGCGGATGG GTGAGTAACACGTAGGTAA CCTGCCCTAAAGCGGGGGATACCATCTGGAAACAGGTG CTAATACCGCATAAACCCA GCAGTCACATGAGTGCTGGTTGAAAGACGGCTTCGGCT GTCACTTTAGGATGGACCT GCGGCGTATTAGCTAGTTGGTGGAGTAACGGTTCACCA AGGCAATGATACGTAGCCG ACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACAC GGCCCAAACTCCTACGGGA GGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCT GATGGAGCAACGCCGCGT GGATGAAGAAGGTCTTCGGATCGTAAAATCCTGTTGTTG AAGAAGAACGGTTGTGAGA GTAACTGCTCATAACGTGACGGTAATCAACCAGAAAGT CACGGCTAACTACGTGCCA GCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGG ATTTATTGGGCGTAAAGGG AGCGCAGGCGGTCTTTTAAGTCTGAATGTGAAAGCCCT CAGCTTAACTGAGGAAGAG CATCGGAAACTGAGAGACTTGAGTGCAGAAGAGGAGAG TGGAACTCCATGTGTAGCG GTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGA AGGCGGCTCTCTGGTCTGT TACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAG GATTAGATACCCTGGTAGT CCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTT TCCGCCTCTCAGTGCTGCA GCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACC GCAAGGTTGAAACTCAAAG GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCAACGC GAAGAACCTTACCAGGTCTTGACATCTCCTGCAAGCCT AAGAGATTAGGGGTTCCCT TCGGGGACAGGAAGACAGGTGGTGCATGGTTGTCGTC AGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTA GTTGCCAGCATTAAGTTGG GCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAG GTGGGGACGACGTCAAATC ATCATGCCCCTTATGACCTGGGCTACACACGTGCTACA ATGGATGGTACAATGAGAA GCGAACTCGCGAGGGGAAGCTGATCTCTGAAAACCATT CTCAGTTCGGATTGCAGGC TGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATC GCGGATCAGCATGCCGCG GTGAATACGTTCCCGGGCCTTGTACACACCGCCC (SEQ ID NO: 38) Silk worm Enterococcus Bombyx mori GutAGGTGATCCAGCCGCACCT TCCGATACGGCTACCTTGT TACGACTTCACCCCAATCATCTATCCCACCTTAGGCGGC TGGCTCCAAAAAGGTTACC TCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGAC GGGCGGTGTGTACAAGGC CCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATT ACTAGCGATTCCGGCTTCA TGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAGAA GCTTTAAGAGATTTGCATGA CCTCGCGGTCTAGCGACTCGTTGTACTTCCCATTGTAGC ACGTGTGTAGCCCAGGTCA TAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCT CCGGTTTGTCACCGGCAGT CTCGCTAGAGTGCCCAACTAAATGATGGCAACTAACAAT AAGGGTTGCGCTCGTTGCG GGACTTAACCCAACATCTCACGACACGAGCTGACGACA ACCATGCACCACCTGTCAC TTTGTCCCCGAAGGGAAAGCTCTATCTCTAGAGTGGTCA AAGGATGTCAAGACCTGGT AAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTC CACCGCTTGTGCGGGCCCC CGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCC CCAGGCGGAGTGCTTAATG CGTTTGCTGCAGCACTGAAGGGCGGAAACCCTCCAACA CTTAGCACTCATCGTTTACG GCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCA CGCTTTCGAGCCTCAGCGT CAGTTACAGACCAGAGAGCCGCCTTCGCCACTGGTGTT CCTCCATATATCTACGCATT TCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCAC TCAAGTCTCCCAGTTTCCAA TGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGA CTTAAGAAACCGCCTGCGC TCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACC TACGTATTACCGCGGCTGC TGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGATACCGT CAGGGGACGTTCAGTTACT AACGTCCTTGTTCTTCTCTAACAACAGAGTTTTACGATCC GAAAACCTTCTTCACTCACG CGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGAT TCCCTACTGCTGCCTCCCG TAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGA TCACCCTCTCAGGTCGGCT ATGCATCGTGGCCTTGGTGAGCCGTTACCTCACCAACT AGCTAATGCACCGCGGGTC CATCCATCAGCGACACCCGAAAGCGCCTTTCACTCTTAT GCCATGCGGCATAAACTGT TATGCGGTATTAGCACCTGTTTCCAAGTGTTATCCCCCT CTGATGGGTAGGTTACCCA CGTGTTACTCACCCGTCCGCCACTCCTCTTTCCAATTGA GTGCAAGCACTCGGGAGGA AAGAAGCGTTCGACTTGCATGTATTAGGCACGCCGCCA GCGTTCGTCCTGAGCCAGG ATCAAACTCT (SEQ ID NO: 39)Delftia Bombyx mori Gut CAGAAAGGAGGTGATCCAG CCGCACCTTCCGATACGGCTACCTTGTTACGACTTCACC CCAGTCACGAACCCCGCCG TGGTAAGCGCCCTCCTTGCGGTTAGGCTACCTACTTCT GGCGAGACCCGCTCCCATG GTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATT CACCGCGGCATGCTGATCC GCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTG CAGACTGCGATCCGGACTA CGACTGGTTTTATGGGATTAGCTCCCCCTCGCGGGTTGG CAACCCTCTGTACCAGCCA TTGTATGACGTGTGTAGCCCCACCTATAAGGGCCATGA GGACTTGACGTCATCCCCA CCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTG CTCAACTGAATGTAGCAACT AATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAA CATCTCACGACACGAGCTG ACGACAGCCATGCAGCACCTGTGTGCAGGTTCTCTTTC GAGCACGAATCCATCTCTG GAAACTTCCTGCCATGTCAAAGGTGGGTAAGGTTTTTC GCGTTGCATCGAATTAAAC CACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCC TTTGAGTTTCAACCTTGCGG CCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTTC GTTACTGAGAAAACTAATTC CCAACAACCAGTTGACATCGTTTAGGGCGTGGACTACC AGGGTATCTAATCCTGTTTG CTCCCCACGCTTTCGTGCATGAGCGTCAGTACAGGTCC AGGGGATTGCCTTCGCCAT CGGTGTTCCTCCGCATATCTACGCATTTCACTGCTACAC GCGGAATTCCATCCCCCTC TACCGTACTCTAGCCATGCAGTCACAAATGCAGTTCCC AGGTTGAGCCCGGGGATTT CACATCTGTCTTACATAACCGCCTGCGCACGCTTTACGC CCAGTAATTCCGATTAACG CTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTA GTTAGCCGGTGCTTATTCTT ACGGTACCGTCATGGGCCCCCTGTATTAGAAGGAGCTTT TTCGTTCCGTACAAAAGCA GTTTACAACCCGAAGGCCTTCATCCTGCACGCGGCATT GCTGGATCAGGCTTTCGCC CATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGT CTGGGCCGTGTCTCAGTCC CAGTGTGGCTGGTCGTCCTCTCAGACCAGCTACAGATC GTCGGCTTGGTAAGCTTTT ATCCCACCAACTACCTAATCTGCCATCGGCCGCTCCAAT CGCGCGAGGCCCGAAGGG CCCCCGCTTTCATCCTCAGATCGTATGCGGTATTAGCTA CTCTTTCGAGTAGTTATCCC CCACGACTGGGCACGTTCCGATGTATTACTCACCCGTTC GCCACTCGTCAGCGTCCGA AGACCTGTTACCGTTCGACTTGCATGTGTAAGGCATGC CGCCAGCGTTCAATCTGAG CCAGGATCAAACTCTACAG TTCGATCT(SEQ ID NO: 40) Pelomonas Bombyx mori Gut ATCCTGGCTCAGATTGAACGCTGGCGGCATGCCTTACA CATGCAAGTCGAACGGTAA CAGGTTAAGCTGACGAGTGGCGAACGGGTGAGTAATAT ATCGGAACGTGCCCAGTCG TGGGGGATAACTGCTCGAAAGAGCAGCTAATACCGCAT ACGACCTGAGGGTGAAAGC GGGGGATCGCAAGACCTCGCNNGATTGGAGCGGCCG ATATCAGATTAGGTAGTTGG TGGGGTAAAGGCCCACCAAGCCAACGATCTGTAGCTGG TCTGAGAGGACGACCAGCC ACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG GCAGCAGTGGGGAATTTTG GACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGC GGGAAGAAGGCCTTCGGGT TGTAAACCGCTTTTGTCAGGGAAGAAAAGGTTCTGGTT AATACCTGGGACTCATGAC GGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAG GGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGT GCGCAGGCGGTTATGCAAG ACAGAGGTGAAATCCCCGGGCTCAACCTGGGAACTGCC TTTGTGACTGCATAGCTAGA GTACGGTAGAGGGGGATGGAATTCCGCGTGTAGCAGT GAAATGCGTAGATATGCGG AGGAACACCGATGGCGAAGGCAATCCCCTGGACCTGTA CTGACGCTCATGCACGAAA GCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC CACGCCCTAAACGATGTCA ACTGGTTGTTGGGAGGGTTTCTTCTCAGTAACGTANNTA ACGCGTGAAGTTGACCGCC TGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAAT TGACGGGGACCCGCACAA GCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAA AACCTTACCTACCCTTGACA TGCCAGGAATCCTGAAGAGATTTGGGAGTGCTCGAAAG AGAACCTGGACACAGGTGC TGCATGGCCGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCG CAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTAAT GAGACTGCCGGTGACAAAC CGGAGGAAGGTGGGGATGACGTCAGGTCATCATGGCC CTTATGGGTAGGGCTACAC ACGTCATACAATGGCCGGGACAGAGGGCTGCCAACCCG CGAGGGGGAGCTAATCCCA GAAACCCGGTCGTAGTCCGGATCGTAGTCTGCAACTCG ACTGCGTGAAGTCGGAATC GCTAGTAATCGCGGATCAGCTTGCCGCGGTGAATACGT TCCCGGGTCTTGTACACAC CGCCCGTCACACCATGGGAGCGGGTTCTGCCAGAAGTA GTTAGCCTAACCGCAAGGA GGGCGATTACCACGGCAGGGTTCGTGACTGGGGTGAA GTCGTAACAAGGTAGCCGT ATCGGAAGGTGCGGCTGGA TCAC (SEQ IDNO: 41)

Any number of bacterial species may be targeted by the compositions ormethods described herein. For example, in some instances, the modulatingagent may target a single bacterial species. In some instances, themodulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500,or more distinct bacterial species. In some instances, the modulatingagent may target any one of about 1 to about 5, about 5 to about 10,about 10 to about 20, about 20 to about 50, about 50 to about 100, about100 to about 200, about 200 to about 500, about 10 to about 50, about 5to about 20, or about 10 to about 100 distinct bacterial species. Insome instances, the modulating agent may target at least about any of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore phyla, classes, orders, families, or genera of bacteria.

In some instances, the modulating agent may increase a population of oneor more bacteria by at least about any of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or more in the host. In some instances, the modulatingagent may reduce the population of one or more bacteria by at leastabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in thehost. In some instances, the modulating agent may eradicate thepopulation of a bacterium in the host.

In some instances, the modulating agent may alter the bacterialdiversity and/or bacterial composition of the host in comparison to ahost organism to which the modulating agent has not been administered.In some instances, the modulating agent may increase the bacterialdiversity in the host relative to a starting diversity by at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the modulating agent may decrease thebacterial diversity in the host relative to a starting diversity by atleast about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or morein comparison to a host organism to which the modulating agent has notbeen administered.

In some instances, the modulating agent may alter the function,activity, growth, and/or division of one or more bacterial cells incomparison to a host organism to which the modulating agent has not beenadministered. For example, the modulating agent may alter the expressionof one or genes in the bacteria. In some instances, the modulating agentmay alter the function of one or more proteins in the bacteria. In someinstances, the modulating agent may alter the function of one or morecellular structures (e.g., the cell wall, the outer or inner membrane)in the bacteria. In some instances, the modulating agent may kill (e.g.,lyse) the bacteria.

The target bacterium may reside in one or more parts of the invertebratehost (e.g., insect, mollusk, or nematode). Further, the target bacteriamay be intracellular or extracellular. In some instances, the bacteriareside in or on one or more parts of the host gut, including, e.g., theforegut, midgut, and/or hindgut. In some instances, the bacteria resideas an intracellular bacteria within a cell of the host. In someinstances, the bacteria reside in a bacteriocyte of the hostinvertebrate (e.g., insect, mollusk, or nematode).

Changes to the populations of bacteria in the host invertebrate (e.g.,insect, mollusk, or nematode) may be determined by any methods known inthe art, such as standard culturing techniques, CFU counts, microarray,polymerase chain reaction (PCR), real-time PCR, flow cytometry,fluorescence microscopy, transmission electron microscopy, fluorescencein situ hybridization (e.g., FISH), spectrophotometry, matrix-assistedlaser desorption ionization-mass spectrometry (MALDI-MS), or DNAsequencing. In some instances, a sample of the host treated with amodulating agent is sequenced (e.g., by metagenomics sequencing of 16SrRNA or rDNA) to determine the microbiome of the host after delivery oradministration of the modulating agent. In some instances, a sample of ahost that did not receive the modulating agent is also sequenced toprovide a reference.

ii. Fungi and Yeast

Exemplary fungi that may be targeted in accordance with the methods andcompositions provided herein, include, but are not limited toAmylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate,Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamycesbromatificus. Non-limiting examples of yeast and yeast-like symbiontsfound in invertebrates (e.g., insect, mollusk, or nematode) includeCandida, Metschnikowia, Leucocoprinu (e.g., Leucocoprinusgongylophorus), Debaromyces, Scheffersomyces shehatae andScheffersomyces stipites, Starmerella, Pichia, Trichosporon,Cryptococcus, Pseudozyma, and yeast-like symbionts from the subphylumPezizomycotina (e.g., Symbiotaphrina bucneri and Symbiotaphrina kochii).Non-limiting examples of yeast that may be targeted by the methods andcompositions herein are listed in Table 2.

TABLE 2 Examples of Yeast in Insects Insect Species Order: Family YeastLocation (Species) Stegobium paniceum Coleoptera: Anobiidae Mycetomes(=Sitodrepa panicea) (Saccharomyces) Cecae (Torulopsis buchnerii)Mycetome between foregut and midgut Mycetomes (Symbiotaphrina buchnerii)Mycetomes and digestive tube (Torulopsis buchnerii) Gut cecae(Symbiotaphrina buchnerii) Lasioderma serricorne Coleoptera: AnobiidaeMycetome between foregut and midgut (Symbiotaphrina kochii) Ernobiusabietis Coleoptera: Anobiidae Mycetomes (Torulopsis karawaiewii)(Candida karawaiewii) Ernobius mollis Coleoptera: Anobiidae Mycetomes(Torulopsis ernobii) (Candida ernobii) Hemicoelus gibbicollisColeoptera: Anobiidae Larval mycetomes Xestobium plumbeum Coleoptera:Anobiidae Mycetomes (Torulopsis xestobii) (Candida xestobii)Criocephalus rusticus Coleoptera: Cerambycidae Mycetomes PhoracanthaColeoptera: Cerambycidae Alimentary canal (Candida semipunctataguilliermondii, C. tenuis) Cecae around midgut (Candida guilliermondii)Harpium inquisitor Coleoptera: Cerambycidae Mycetomes (Candida rhagii)Harpium mordax Coleoptera: Cerambycidae Cecae around midgut (Candida H.sycophanta tenuis) Gaurotes virginea Coleoptera: Cerambycidae Cecaearound midgut (Candida rhagii) Leptura rubra Coleoptera: CerambycidaeCecae around midgut (Candida tenuis) Cecae around midgut (Candidaparapsilosis) Leptura maculicornis Coleoptera: Cerambycidae Cecae aroundmidgut (Candida parapsilosis) L. cerambyciformis Leptura sanguinolentaColeoptera: Cerambycidae Cecae around midgut (Candida sp.) Rhagiumbifasciatum Coleoptera: Cerambycidae Cecae around midgut (Candidatenuis) Rhagium inquisitor Coleoptera: Cerambycidae Cecae around midgut(Candida guilliermondii) Rhagium mordax Coleoptera: Cerambycidae Cecaearound midgut (Candida) Carpophilus Coleoptera: Nitidulidae Intestinaltract (10 yeast species) hemipterus Odontotaenius Coleoptera: PassalidaeHindgut (Enteroramus dimorphus) disjunctus Odontotaenius Coleoptera:Passalidae Gut (Pichia stipitis, P. segobiensis, disjunctus Candidashehatae) Verres sternbergianus (C. ergatensis) Scarabaeus Coleoptera:Scarabaeidae Digestive tract (10 yeast species) semipunctatus Chironitisfurcifer Unknown species Coleoptera: Scarabaeidae Guts (Trichosporoncutaneum) Dendroctonus and Ips Coleoptera: Scolytidae Alimentary canal(13 yeast spp. species) Dendroctonus frontalis Coleoptera: ScolytidaeMidgut (Candida sp.) Ips sexdentatus Coleoptera: Scolytidae Digestivetract (Pichia bovis, P. rhodanensis) Hansenula holstii (Candida rhagii)Digestive tract (Candida pulcherina) Ips typographus Coleoptera:Scolytidae Alimentary canal Alimentary tracts (Hansenula capsulata,Candida parapsilosis) Guts and beetle homogenates (Hansenula holstii, H.capsulata, Candida diddensii, C. mohschtana, C. nitratophila,Cryptococcus albidus, C. laurentii) Trypodendron Coleoptera: ScolytidaeNot specified lineatum Xyloterinus politus Coleoptera: Scolytidae Head,thorax, abdomen (Candida, Pichia, Saccharomycopsis) Periplanetaamericana Dictyoptera: Blattidae Hemocoel (Candida sp. nov.) Blattaorientalis Dictyoptera: Blattidae Intestinal tract (Kluyveromycesblattae) Blatella germanica Dictyoptera: Blattellidae HemocoelCryptocercus Dictyoptera: Cryptocercidae Hindgut (1 yeast species)punctulatus Philophylla heraclei Diptera: Tephritidae Hemocoel Aedes (4species) Diptera: Culicidae Internal microflora (9 yeast genera)Drosophila Diptera: Drosophilidae Alimentary canal (24 yeastpseudoobscura species) Drosophila (5 spp.) Diptera: Drosophilidae Crop(42 yeast species) Drosophila Diptera: Drosophilidae Crop (8 yeastspecies) melanogaster Drosophila (4 spp.) Diptera: Drosophilidae Crop (7yeast species) Drosophila (6 spp.) Diptera: Drosophilidae Larval gut (17yeast species) Drosophila (20 spp.) Diptera: Drosophilidae Crop (20yeast species) Drosophila (8 species Diptera: Drosophilidae Crop(Kloeckera, Candida, groups) Kluyveromyces) Drosophila serido Diptera:Drosophilidae Crop (18 yeast species) Drosophila (6 spp.) Diptera:Drosophilidae Intestinal epithelium (Coccidiascus legeri) ProtaxymiaDiptera Unknown (Candida, Cryptococcus, melanoptera Sporoblomyces)Astegopteryx styraci Homoptera: Aphididae Hemocoel and fat bodyTuberaphis sp. Homoptera: Aphididae Tissue sections Hamiltonaphisstyraci Glyphinaphis bambusae Cerataphis sp. Hamiltonaphis styraciHomoptera: Aphididae Abdominal hemocoel Cofana unimaculata Homoptera:Cicadellidae Fat body Leofa unicolor Homoptera: Cicadellidae Fat bodyLecaniines, etc. Homoptera: Coccoidea d Hemolymph, fatty tissue, etc.Lecanium sp. Homoptera: Coccidae Hemolymph, adipose tissue Ceroplastes(4 sp.) Homoptera: Coccidae Blood smears Laodelphax striatellusHomoptera: Delphacidae Fat body Eggs Eggs (Candida) Nilaparvata lugensHomoptera: Delphacidae Fat body Eggs (2 unidentified yeast species)Eggs, nymphs (Candida) Eggs (7 unidentified yeast species) Eggs(Candida) Nisia nervosa Homoptera: Delphacidae Fat body Nisia grandicepsPerkinsiella spp. Sardia rostrata Sogatella furcifera Sogatodesorizicola Homoptera: Delphacidae Fat body Amrasca devastans Homoptera:Jassidae Eggs, mycetomes, hemolymph Tachardina lobata Homoptera:Kerriidae Blood smears (Torulopsis) Laccifer (=Lakshadia) Homoptera:Kerriidae Blood smears (Torula variabilis) sp. Comperia mercetiHymenoptera: Encyrtidae Hemolymph, gut, poison gland Solenopsis invictaHymenoptera: Formicidae Hemolymph (Myrmecomyces annellisae) S.quinquecuspis Solenopsis invicta Hymenoptera: Formicidae Fourth instarlarvae (Candida parapsilosis, Yarrowia lipolytica) Gut and hemolymph(Candida parapsilosis, C. lipolytica, C. guillermondii, C. rugosa,Debaryomyces hansenii) Apis mellifera Hymenoptera: Apidae Digestivetracts (Torulopsis sp.) Intestinal tract (Torulopsis apicola) Digestivetracts (8 yeast species) Intestinal contents (12 yeast species)Intestinal contents (7 yeast species) Intestines (14 yeast species)Intestinal tract (Pichia melissophila) Intestinal tracts (7 yeastspecies) Alimentary canal (Hansenula silvicola) Crop and gut (13 yeastspecies) Apis mellifera Hymenoptera: Apidae Midguts (9 yeast genera)Anthophora Hymenoptera: Anthophoridae occidentalis Nomia melanderiHymenoptera: Halictidae Halictus rubicundus Hymenoptera: HalictidaeMegachile rotundata Hymenoptera: Megachilidae

Any number of fungal species may be targeted by the compositions ormethods described herein. For example, in some instances, the modulatingagent may target a single fungal species. In some instances, themodulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500,or more distinct fungal species. In some instances, the modulating agentmay target any one of about 1 to about 5, about 5 to about 10, about 10to about 20, about 20 to about 50, about 50 to about 100, about 100 toabout 200, about 200 to about 500, about 10 to about 50, about 5 toabout 20, or about 10 to about 100 distinct fungal species. In someinstances, the modulating agent may target at least about any of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore phyla, classes, orders, families, or genera of fungi.

In some instances, the modulating agent may increase a population of oneor more fungi by at least about any of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or more in the host in comparison to a host organism towhich the modulating agent has not been administered. In some instances,the modulating agent may reduce the population of one or more fungi byat least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore in the host in comparison to a host organism to which themodulating agent has not been administered. In some instances, themodulating agent may eradicate the population of a fungi in the host.

In some instances, the modulating agent may alter the fungal diversityand/or fungal composition of the host. In some instances, the modulatingagent may increase the fungal diversity in the host relative to astarting diversity by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more in comparison to a host organism to whichthe modulating agent has not been administered. In some instances, themodulating agent may decrease the fungal diversity in the host relativeto a starting diversity by at least about any of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism towhich the modulating agent has not been administered.

In some instances, the modulating agent may alter the function,activity, growth, and/or division of one or more fungi. For example, themodulating agent may alter the expression of one or more genes in thefungus. In some instances, the modulating agent may alter the functionof one or more proteins in the fungus. In some instances, the modulatingagent may alter the function of one or more cellular components in thefungus. In some instances, the modulating agent may kill the fungus.

Further, the target fungus may reside in one or more parts of theinsect. In some instances, the fungus resides in or on one or more partsof the insect gut, including, e.g., the foregut, midgut, and/or hindgut.In some instances, the fungus lives extracellularly in the hemolymph,fat bodies or in specialized structures in the host.

Changes to the population of fungi in the host may be determined by anymethods known in the art, such as microarray, polymerase chain reaction(PCR), real-time PCR, flow cytometry, fluorescence microscopy,transmission electron microscopy, fluorescence in situ hybridization(e.g., FISH), spectrophotometry, matrix-assisted laser desorptionionization-mass spectrometry (MALDI-MS), and DNA sequencing. In someinstances, a sample of the host treated with a modulating agent issequenced (e.g., by metagenomics sequencing) to determine the microbiomeof the host after delivery or administration of the modulating agent. Insome instances, a sample of a host that did not receive the modulatingagent is also sequenced to provide a reference.

III. Modulating Agents

The modulating agent of the methods and compositions provided herein mayinclude a polypeptide, a nucleic acid, small molecule, or anycombination thereof. In some instances, the modulating agent is anucleic acid molecule (e.g., DNA molecule or RNA molecule, e.g., mRNA,guide

RNA (gRNA), or inhibitory RNA molecule (e.g., siRNA, shRNA, or miRNA),or a hybrid DNA-RNA molecule), a small molecule, a peptide, or apolypeptide (e.g., an antibody molecule, e.g., an antibody or antigenbinding fragment thereof). Any of these agents can be used to alter themicrobiota of a host invertebrate (e.g., insect, mollusk, or nematode)by targeting pathways in the host and/or resident microorganisms (e.g.,pathways that mediate host-microbiota interactions, e.g., host immunesystem pathways or bacteriocyte pathways). For example, any modulatingagents described herein may be used to regulate (e.g., to induce or toinhibit) a gene or protein in the host or a microorganism resident inthe host (e.g., a protein or a gene encoding a protein listed in Table7, Table 8, or Table 9).

i. Polypeptides

The modulating agent described herein may include a polypeptide (e.g.,antibody). In some instances, the modulating agent described hereinincludes a polypeptide or functional fragments or derivative thereof,which target pathways in the host invertebrate (e.g., insect, mollusk,or nematode) and/or resident microorganisms (e.g., pathways that mediatehost-microbiota interactions, e.g., host immune system pathways orbacteriocyte pathways). In some instances, the agent is a polypeptidelisted in Table 7, Table 8, or Table 9, wherein the primary sequence ofthe agent polypeptide is provided by reference to its accession number.

A modulating agent including a polypeptide as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of concentration inside a target host; (b) reach a target level (e.g., apredetermined or threshold level) of concentration inside a target hostgut; (c) reach a target level (e.g., a predetermined or threshold level)of concentration inside a target host bacteriocyte; (d) modulate thelevel, or an activity, of one or more microorganism (e.g., endosymbiont)in the target host; or/and (e) modulate fitness of the target host.

Polypeptides included herein may include naturally occurringpolypeptides or recombinantly produced variants. In some instances, thepolypeptide may be a functional fragments or variants thereof (e.g., anenzymatically active fragment or variant thereof of polypeptide listedin Table 7, Table 8, or Table 9). Such fragments or variants can be madeand screened for similar activity as described herein and would beequivalent hereunder in the methods and compositions disclosed). Forexample, the polypeptide may be a functionally active variant of any ofthe polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g.,over a specified region or over the entire sequence, to a sequence of apolypeptide described herein or a naturally occurring polypeptide. Insome instances, the polypeptide may have at least 50% (e.g., at least50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to theamino acid sequence of a protein listed in Table 7, Table 8, or Table 9with reference to the accession number provided.

Methods of making a therapeutic polypeptide are routine in the art. See,in general, Smales & James (Eds.), Therapeutic Proteins: Methods andProtocols (Methods in Molecular Biology), Humana Press (2005); andCrommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology:Fundamentals and Applications, Springer (2013).

Methods for producing a polypeptide involve expression in mammaliancells, although recombinant proteins can also be produced using insectcells, yeast, bacteria, or other cells under the control of appropriatepromoters. Mammalian expression vectors may comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer, and other 5′ or 3′ flanking nontranscribed sequences, and 5′or 3′ nontranslated sequences such as necessary ribosome binding sites,a polyadenylation site, splice donor and acceptor sites, and terminationsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described in Green & Sambrook,Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold SpringHarbor Laboratory Press (2012).

Various mammalian cell culture systems can be employed to express andmanufacture a recombinant polypeptide agent (e.g., listed in Tables 4,5, or 6). Examples of mammalian expression systems include CHO cells,COS cells, HeLA and BHK cell lines. Processes of host cell culture forproduction of protein therapeutics are described in, e.g., Zhou andKantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing(Advances in Biochemical Engineering/Biotechnology), Springer (2014).Purification of protein therapeutics is described in Franks, ProteinBiotechnology: Isolation, Characterization, and Stabilization, HumanaPress (2013); and in Cutler, Protein Purification Protocols (Methods inMolecular Biology), Humana Press (2010). Formulation of proteintherapeutics is described in Meyer (Ed.), Therapeutic Protein DrugProducts: Practical Approaches to formulation in the Laboratory,Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

The polypeptide modulating agents discussed hereinafter, namelyantibodies, bacteriocins, antimicrobial peptides, and bacteriocyteregulatory peptides, can be used to alter pathways in the host thatmediate interactions between the host and microorganisms resident in thehost as indicated in the sections for increasing or decreasing thefitness of hosts.

(a) Antibodies

In some instances, the modulating agent includes an antibody or antigenbinding fragment thereof. For example, an agent described herein may bean antibody that blocks or potentiates activity and/or function of acomponent of the host immune system pathway or bacteriocyte regulatorypathway listed in Table 8 or Table 9. The antibody may act as anantagonist or agonist of a polypeptide (e.g., enzyme or cell receptor)in the host or microorganisms resident in the host, including anyproteins list in Table 7, Table 8, or Table 9.

The making and use of antibodies against a target antigen (e.g.,proteins that mediate host-microbiota interactions, e.g., host immunesystem proteins or bacteriocyte proteins, e.g., proteins in Table 7,Table 8, or Table 9) is known in the art. See, for example, Zhiqiang An(Ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, 1stEdition, Wiley, 2009 and also Greenfield (Ed.), Antibodies: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press, 2013, formethods of making recombinant antibodies, including antibodyengineering, use of degenerate oligonucleotides, 5′-RACE, phage display,and mutagenesis; antibody testing and characterization; antibodypharmacokinetics and pharmacodynamics; antibody purification andstorage; and screening and labeling techniques.

(b) Bacteriocins

The modulating agent described herein may include a bacteriocin. In someinstances, the bacteriocin is naturally produced by Gram-positivebacteria, such as Pseudomonas, Streptomyces, Bacillus, Staphylococcus,or lactic acid bacteria (LAB, such as Lactococcus lactis). In someinstances, the bacteriocin is naturally produced by Gram-negativebacteria, such as Hafnia alvei, Citrobacter freundii, Klebsiellaoxytoca, Klebsiella pneumonia, Enterobacter cloacae, Serratiaplymithicum, Xanthomonas campestris, Erwinia carotovora, Ralstoniasolanacearum, or Escherichia coli. Exemplary bacteriocins include, butare not limited to, Class I-IV LAB antibiotics (such as lantibiotics),colicins, microcins, and pyocins. Non-limiting examples of bacteriocinsare listed in Table 3.

TABLE 3 Examples of Bacteriocins Class Name Producer Targets SequenceClass I Nisin Lactococcus Active on Gram-positive ITSISLCTPGCKT lactisbacteria: GALMGCNMKTA Enterococcus, TCHCSIHVSK Lactobacillus, (SEQ IDNO: 42) Lactococcus, Leuconostoc, Listeria, Clostridium EpiderminStaphylococcus Gram-positive bacteria IASKFICTPGCAK epidermis TGSFNSYCC(SEQ ID NO: 43) Class II Class II a Pediocin Pediococcus Pediococci,KYYGNGVTCG PA-1 acidilactici Lactobacilli, KHSCSVDWGK Leuconostoc,ATTCIINNGAMA Brochothrix WATGGHQGNHKC thermosphacta, (SEQ ID NO: 44)Propionibacteria, Bacilli, Enterococci, Staphylococci, Listeriaclostridia, Listeria monocytogenes, Listeria innocua Class II bEnterocin P Enterococcus Lactobacillus sakei, ATRSYGNGVYC faeciumEnterococcus faecium NNSKCWVNWGE AKENIAGIVISGW ASGLAGMGH (SEQ ID NO: 45)Class II c Lactococcin G Streptococcus Gram-positive bacteriaGTWDDIGQGIGR lactis VAYWVGKAMGN MSDVNQASRINR KKKH (SEQ ID NO: 46) ClassII d Lactacin-F Lactobacillus Lactobacilli, NRWGDTVLSAA johnsoniiEnterococcus faecalis SGAGTGIKACKS FGPWGMAICGV GGAAIGGYFGYT HN (SEQ IDNO: 47) Class III Class III a Enterocin Enterococcus Broad spectrum:Gram MAKEFGIPAAVA AS-48 faecalis positive and Gram GTVLNVVEAGG negativebacteria. WVTTIVSILTAVG SGGLSLLAAAGR ESIKAYLKKEIKK KGKRAVIAW (SEQ ID NO:48) Class III b Aureocin Staphylococcus Broad spectrum: GramMSWLNFLKYIAK A70 aureus positive and Gram YGKKAVSAAWK negative bacteria.YKGKVLEWLNV GPTLEWVWQKL KKIAGL (SEQ ID NO: 49) Class IV Garvicin ALactococcus Broad spectrum: Gram IGGALGNALNGL garvieae positive and GramGTWANMMNGG negative bacteria. GFVNQWQVYAN KGKINQYRPY (SEQ ID NO: 50)Unclassified Colicin V Escherichia Active against MRTLTLNELDSV coliEscherichia coli (also SGGASGRDIAMA closely related bacteria),IGTLSGQFVAGGI Enterobacteriaceae GAAAGGVAGGAI YDYASTHKPNPA MSPSGLGGTIKQKPEGIPSEAWNY AAGRLCNWSPN NLSDVCL (SEQ ID NO: 51)

In some instances, the bacteriocin is a colicin, a pyocin, or a microcinproduced by Gram-negative bacteria. In some instances, the bacteriocinis a colicin. The colicin may be a group A colicin (e.g., uses the Tolsystem to penetrate the outer membrane of a target bacterium) or a groupB colicin (e.g., uses the Ton system to penetrate the outer membrane ofa target bacterium). In some instances, the bacteriocin is a microcin.The microcin may be a class I microcin (e.g., <5 kDa, haspost-translational modifications) or a class II microcin (e.g., 5-10kDa, with or without post-translational modifications). In someinstances, the class II microcin is a class IIa microcin (e.g., requiresmore than one genes to synthesize and assemble functional peptides) or aclass IIb microcin (e.g., linear peptides with or withoutpost-translational modifications at C-terminus). In some instances, thebacteriocin is a pyocin. In some instances, the pyocin is an R-pyocin,F-pyocin, or S-pyocin.

In some instances, the bacteriocin is a class I, class II, class III, orclass IV bacteriocin produced by Gram-positive bacteria. In someinstances, the modulating agent includes a Class I bacteriocin (e.g.,lanthionine-containing antibiotics (lantibiotics) produced by aGram-positive bacteria). The class I bacteriocins or lantibiotic may bea low molecular weight peptide (e.g., less than about 5 kDa) and maypossess post-translationally modified amino acid residues (e.g.,lanthionine, β-methyllanthionine, or dehydrated amino acids).

In some instances, the bacteriocin is a Class II bacteriocin (e.g.,non-lantibiotics produced by Gram-positive bacteria). Many arepositively charged, non-lanthionine-containing peptides, which unlikelantibiotics, do not undergo extensive post-translational modification.The Class II bacteriocin may belong to one of the following subclasses:“pediocin-like” bacteriocins (e.g., pediocin PA-1 and carnobacteriocin X(Class IIa)); two-peptide bacteriocins (e.g., lactacin F and ABP-118(Class IIb)); circular bacteriocins (e.g., carnocyclin A and andenterocin AS-48 (Class 11c)); or unmodified, linear, non-pediocin-likebacteriocins (e.g., epidermicin NI01 and lactococcin A (Class Ild)).

In some instances, the bacteriocin is a Class III bacteriocin (e.g.,produced by Gram-positive bacteria). Class III bacteriocins may have amolecular weight greater than 10 kDa and may be heat unstable proteins.The Class III bacteriocins can be further subdivided into Group IIIA andGroup IIIB bacteriocins. The Group IIIA bacteriocins includebacteriolytic enzymes which kill sensitive strains by lysis of the cellwell, such as Enterolisin A. Group IIIB bacteriocins include non-lyticproteins, such as Caseicin 80, Helveticin J, and lactacin B.

In some instances, the bacteriocin is a Class IV bacteriocin (e.g.,produced by Gram-positive bacteria). Class IV bacteriocins are a groupof complex proteins, associated with other lipid or carbohydratemoieties, which appear to be required for activity. They are relativelyhydrophobic and heat stable. Examples of Class IV bacteriocinsleuconocin S, lactocin 27, and lactocin S.

In some instances, the bacteriocin is an R-type bacteriocin. R-typebacteriocins are contractile bacteriocidal protein complexes. SomeR-type bacteriocins have a contractile phage-tail-like structure. TheC-terminal region of the phage tail fiber protein determinestarget-binding specificity. They may attach to target cells through areceptor-binding protein, e.g., a tail fiber. Attachment is followed bysheath contraction and insertion of the core through the envelope of thetarget bacterium. The core penetration results in a rapid depolarizationof the cell membrane potential and prompt cell death. Contact with asingle R-type bacteriocin particle can result in cell death. An R-typebacteriocin, for example, may be thermolabile, mild acid resistant,trypsin resistant, sedimentable by centrifugation, resolvable byelectron microscopy, or a combination thereof. Other R-type bacteriocinsmay be complex molecules including multiple proteins, polypeptides, orsubunits, and may resemble a tail structure of bacteriophages of themyoviridae family. In naturally occurring R-type bacteriocins, thesubunit structures may be encoded by a bacterial genome, such as that ofC. difficile or P. aeruginosa and form R-type bacteriocins to serve asnatural defenses against other bacteria. In some instances, the R-typebacteriocin is a pyocin. In some instances, the pyocin is an R-pyocin,F-pyocin, or S-pyocin.

In some instances, the bacteriocin is a functionally active variant ofthe bacteriocins described herein. In some instances, the variant of thebacteriocin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specifiedregion or over the entire sequence, to a sequence of a bacteriocindescribed herein or a naturally occurring bacteriocin.

In some instances, the bacteriocin may be bioengineered, according tostandard methods, to modulate their bioactivity, e.g., increase ordecrease or regulate, or to specify their target microorganisms. Inother instances, the bacteriocin is produced by the translationalmachinery (e.g. a ribosome, etc.) of a microbial cell. In someinstances, the bacteriocin is chemically synthesized. Some bacteriocinscan be derived from a polypeptide precursor. The polypeptide precursorcan undergo cleavage (e.g., processing by a protease) to yield thepolypeptide of the bacteriocin itself. As such, in some instances, thebacteriocin is produced from a precursor polypeptide. In some otherinstances, the bacteriocin includes a polypeptide that has undergonepost-translational modifications, for example, cleavage, or the additionof one or more functional groups.

The bacteriocins described herein may be formulated in a composition forany of the uses described herein. The compositions disclosed herein mayinclude any number or type (e.g., classes) of bacteriocins, such as atleast about any one of 1 bacteriocin, 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 100, or more bacteriocins. Suitable concentrations of eachbacteriocin in the compositions described herein depends on factors suchas efficacy, stability of the bacteriocin, number of distinctbacteriocin types in the compositions, formulation, and methods ofapplication of the composition. In some instances, each bacteriocin in aliquid composition is from about 0.01 ng/ml to about 100 mg/mL. In someinstances, each bacteriocin in a solid composition is from about 0.01ng/g to about 100 mg/g. In some instances, wherein the compositionincludes at least two types of bacteriocins, the concentration of eachtype of the bacteriocins may be the same or different. In someinstances, the bacteriocin is provided in a composition including abacterial cell that secretes the bacteriocin. In some instances, thebacteriocin is provided in a composition including a polypeptide (e.g.,a polypeptide isolated from a bacterial cell).

Bacteriocins may neutralize (e.g., kill) at least one microorganismother than the individual bacterial cell in which the polypeptide ismade, including cells clonally related to the bacterial cell and othermicrobial cells. As such, a bacterial cell may exert cytotoxic orgrowth-inhibiting effects on a plurality of microbial organisms bysecreting bacteriocins. In some instances, the bacteriocin targets andkills one or more species of bacteria resident in the host viacytoplasmic membrane pore formation, cell wall interference (e.g.,peptidoglycanase activity), or nuclease activity (e.g., DNase activity,16S rRNase activity, or tRNase activity).

In some instances, the bacteriocin has a neutralizing activity.Neutralizing activity of bacteriocins may include, but is not limitedto, arrest of microbial reproduction, or cytotoxicity. Some bacteriocinshave cytotoxic activity, and thus can kill microbial organisms, forexample bacteria, yeast, algae, and the like. Some bacteriocins caninhibit the reproduction of microbial organisms, for example bacteria,yeast, algae, and the like, for example by arresting the cell cycle.

In some instances, the bacteriocin has killing activity. The killingmechanism of bacteriocins is specific to each group of bacteriocins. Insome instances, the bacteriocin has narrow-spectrum bioactivity.Bacteriocins are known for their very high potency against their targetstrains. Some bacteriocin activity is limited to strains that areclosely related to the bacteriocin producer strain (narrow-spectrumbioactivity). In some instances, the bacteriocin has broad-spectrumbioactivity against a wide range of genera.

In some instances, bacteriocins interact with a receptor molecule or adocking molecule on the target bacterial cell membrane. For example,nisin is extremely potent against its target bacterial strains, showingantimicrobial activity even at a single-digit nanomolar concentration.The nisin molecule has been shown to bind to lipid II, which is the maintransporter of peptidoglycan subunits from the cytoplasm to the cellwall

In some instances, the bacteriocin has anti-fungal activity. A number ofbacteriocins with anti-yeast or anti-fungal activity have beenidentified. For example, bacteriocins from Bacillus have been shown tohave neutralizing activity against some yeast strains (see, for example,Adetunji and Olaoye, Malaysian Journal of Microbiology 9:130-13, 2013).In another example, an Enterococcus faecalis peptide has been shown tohave neutralizing activity against Candida species (see, for example,Shekh and Roy, BMC Microbiology 12:132, 2012). In another example,bacteriocins from Pseudomonas have been shown to have neutralizingactivity against fungi, such as Curvularia lunata, Fusarium species,Helminthosporium species, and Biopolaris species (see, for example,Shalani and Srivastava, The Internet Journal of Microbiology Volume 5Number 2, 2008). In another example, botrycidin AJ1316 and alirin B1from B. subtilis have been shown to have antifungal activities.

A modulating agent including a bacteriocin as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of bacteriocin concentration inside a target host; (b) reach a targetlevel (e.g., a predetermined or threshold level) of bacteriocinconcentration inside a target host gut; (c) reach a target level (e.g.,a predetermined or threshold level) of bacteriocin concentration insidea target host bacteriocyte; (d) modulate the level, or an activity, ofone or more microorganism (e.g., endosymbiont) in the target host;or/and (e) modulate fitness of the target host.

As illustrated by Example 5, bacteriocins (e.g., colA produced by atransgenic plant) can be used as a modulating agent that targets a hostpathway (e.g., an insect, e.g., an aphid) that alters the activity,levels, or metabolism of endosymbiotic bacteria resident in the host,such as a Buchnera spp., to modulate (e.g., decrease) the fitness of thehost.

(c) Antimicrobial Peptides

The modulating agent described herein may include an antimicrobialpeptide (AMP). Any AMP suitable for inhibiting a microorganism residentin the host may be used. AMPs are a diverse group of molecules, whichare divided into subgroups on the basis of their amino acid compositionand structure. The AMP may be derived or produced from any organism thatnaturally produces AMPs, including AMPs derived from plants (e.g.,copsin), insects (e.g., mastoparan, poneratoxin, cecropin, moricin,melittin), frogs (e.g., magainin, dermaseptin, aurein), and mammals(e.g., cathelicidins, defensins and protegrins). Non-limiting examplesof AMPs are listed in Table 4.

TABLE 4 Examples of Antimicrobial Peptides Example Type CharacteristicAMP Sequence Anionic rich in glutamic and dermcidinSSLLEKGLDGAKKAVGGLGKL peptides aspartic acid GKDAVEDLESVGKGAVHDVKDVLDSVL (SEQ ID NO: 52) Linear cationic lack cysteine cecropin AKWKLFKKIEKVGQNIRDGIIKAG α-helical PAVAVVGQATQIAK peptides (SEQ ID NO:53) andropin MKYFSVLVVLTLILAIVDQSDAFI NLLDKVEDALHTGAQAGFKLIRPVERGATPKKSEKPEK (SEQ ID NO: 54) moricin MNILKFFFVFIVAMSLVSCSTAAPAKIPIKAIKTVGKAVGKGLRAI NIASTANDVFNFLKPKKRKH (SEQ ID NO: 55) ceratotoxinMANLKAVFLICIVAFIALQCVVA EPAAEDSVVVKRSIGSALKKAL PVAKKIGKIALPIAKAALPVAAGLVG (SEQ ID NO: 56) Cationic rich in proline, arginine, abaecinMKVVIFIFALLATICAAFAYVPLP peptide phenylalanine, glycine,NVPQPGRRPFPTFPGQGPFNP enriched for tryptophan KIKWPQGY specific amino(SEQ ID NO: 57) acid apidaecins KNFALAILVVTFVVAVFGNTNLDPPTRPTRLRREAKPEAEPGNN RPVYIPQPRPPHPRLRREAEPE AEPGNNRPVYIPQPRPPHPRLRREAELEAEPGNNRPVYISQP RPPHPRLRREAEPEAEPGNNR PVYIPQPRPPHPRLRREAELEAEPGNNRPVYISQPRPPHPRLR REAEPEAEPGNNRPVYIPQPR PPHPRLRREAEPEAEPGNNRPVYIPQPRPPHPRLRREAEPEAE PGNNRPVYIPQPRPPHPRLRR EAKPEAKPGNNRPVYIPQPRP PHPRI(SEQ ID NO: 58) prophenin METQRASLCLGRWSLWLLLLA LVVPSASAQALSYREAVLRAVDRLNEQSSEANLYRLLELDQPPK ADEDPGTPKPVSFTVKETVCP RPTRRPPELCDFKENGRVKQCVGTVTLDQIKDPLDITCNEGVR RFPWWWPFLRRPRLRRQAFP PPNVPGPRFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPP NFPGPPFPPPIFPGPWFPPPPP FRPPPFGPPRFPGRR (SEQ ID NO:59) indolicidin MQTQRASLSLGRWSLWLLLLG LVVPSASAQALSYREAVLRAVDQLNELSSEANLYRLLELDPPPK DNEDLGTRKPVSFTVKETVCP RTIQQPAEQCDFKEKGRVKQCVGTVTLDPSNDQFDLNCNELQ SVILPWKWPWWPWRRG (SEQ ID NO: 60) Anionic andcontain 1-3 disulfide bond protegrin METQRASLCLGRWSLWLLLLA cationicLVVPSASAQALSYREAVLRAVD peptides that RLNEQSSEANLYRLLELDQPPK containADEDPGTPKPVSFTVKETVCP cysteine and RPTRQPPELCDFKENGRVKQC form disulfideVGTVTLDQIKDPLDITCNEVQG bonds VRGGRLCYCRRRFCVCVGRG (SEQ ID NO: 61)tachyplesins KWCFRVCYRGICYRRCR (SEQ ID NO: 62) defensinMKCATIVCTIAVVLAATLLNGSV QAAPQEEAALSGGANLNTLLD ELPEETHHAALENYRAKRATCDLASGFGVGSSLCAAHCIARR YRGGYCNSKAVCVCRN (SEQ ID NO: 63) drosomycinMMQIKYLFALFAVLMLVVLGAN EADADCLSGRYKGPCAVWDN ETCRRVCKEEGRSSGHCSPSLKCWCEGC (SEQ ID NO: 64)

The AMP may be active against any number of target microorganisms. Insome instances, the AMP may have antibacterial and/or antifungalactivities. In some instances, the AMP may have a narrow-spectrumbioactivity or a broad-spectrum bioactivity. For example, some AMPstarget and kill only a few species of bacteria or fungi, while othersare active against both gram-negative and gram-positive bacteria as wellas fungi.

Further, the AMP may function through a number of known mechanisms ofaction. For example, the cytoplasmic membrane is a frequent target ofAMPs, but AMPs may also interfere with DNA and protein synthesis,protein folding, and cell wall synthesis. In some instances, AMPs withnet cationic charge and amphipathic nature disrupt bacterial membranesleading to cell lysis. In some instances, AMPs may enter cells andinteract with intracellular target to interfere with DNA, RNA, protein,or cell wall synthesis. In addition to killing microorganisms, AMPs havedemonstrated a number of immunomodulatory functions that are involved inthe clearance of infection, including the ability to alter host geneexpression, act as chemokines and/or induce chemokine production,inhibit lipopolysaccharide induced pro-inflammatory cytokine production,promote wound healing, and modulating the responses of dendritic cellsand cells of the adaptive immune response.

In some instances, the AMP is a functionally active variant of the AMPsdescribed herein. In some instances, the variant of the AMP has at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity, e.g., over a specified region or over the entiresequence, to a sequence of an AMP described herein or a naturallyderived AMP.

In some instances, the AMP may be bioengineered to modulate itsbioactivity, e.g., increase or decrease or regulate, or to specify atarget microorganism. In some instances, the AMP is produced by thetranslational machinery (e.g. a ribosome, etc.) of a cell. In someinstances, the AMP is chemically synthesized. In some instances, the AMPis derived from a polypeptide precursor. The polypeptide precursor canundergo cleavage (for example, processing by a protease) to yield thepolypeptide of the AMP itself. As such, in some instances, the AMP isproduced from a precursor polypeptide. In some instances, the AMPincludes a polypeptide that has undergone post-translationalmodifications, for example, cleavage, or the addition of one or morefunctional groups.

The AMPs described herein may be formulated in a composition for any ofthe uses described herein. The compositions disclosed herein may includeany number or type (e.g., classes) of AMPs, such as at least about anyone of 1 AMP, 2, 3, 4, 5, 10, 15, 20, or more AMPs. A suitableconcentration of each AMP in the composition depends on factors such asefficacy, stability of the AMP, number of distinct AMP in thecomposition, the formulation, and methods of application of thecomposition. In some instances, each AMP in a liquid composition is fromabout 0.1 ng/mL to about 100 mg/mL. In some instances, each AMP in asolid composition is from about 0.1 ng/g to about 100 mg/g. In someinstances, wherein the composition includes at least two types of AMPs,the concentration of each type of AMP may be the same or different.

A modulating agent including an AMP as described herein can be contactedwith the target host in an amount and for a time sufficient to: (a)reach a target level (e.g., a predetermined or threshold level) of AMPconcentration inside a target host; (b) reach a target level (e.g., apredetermined or threshold level) of AMP concentration inside a targethost gut; (c) reach a target level (e.g., a predetermined or thresholdlevel) of AMP concentration inside a target host bacteriocyte; (d)modulate the level, or an activity, of one or more microorganism (e.g.,endosymbiont) in the target host; or/and (e) modulate fitness of thetarget host.

(d) Bacteriocyte Regulatory Peptides

The modulating agent described herein may include a bacteriocyteregulatory peptide (BRP). BRPs are peptides expressed in thebacteriocytes of insects. These genes are expressed first at adevelopmental time point coincident with the incorporation of symbiontsand their bacteriocyte-specific expression is maintained throughout theinsect's life. In some instances, the BRP has a hydrophobic aminoterminal domain, which is predicted to be a signal peptide. In addition,some BRPs have a cysteine-rich domain. In some instances, thebacteriocyte regulatory peptide is a bacteriocyte-specific cysteine rich(BCR) protein. Bacteriocyte regulatory peptides have a length betweenabout 40 and 150 amino acids. In some instances, the bacteriocyteregulatory peptide has a length in the range of about 45 to about 145,about 50 to about 140, about 55 to about 135, about 60 to about 130,about 65 to about 125, about 70 to about 120, about 75 to about 115,about 80 to about 110, about 85 to about 105, or any range therebetween.Non-limiting examples of BRPs and their activities are listed in Table5.

TABLE 5 Examples of Bacteriocyte Regulatory Peptides Name PeptideSequence Bacteriocyte-specific cysteine richMKLLHGFLIIMLTMHLSIQYAYGGPFLTKYLCDRVCHKLC proteins BCR family, peptideBCR1 GDEFVCSCIQYKSLKGLWFPHCPTGKASVVLHNFLTSP (SEQ ID NO: 65)Bacteriocyte-specific cysteine richMKLLYGFLIIMLTIHLSVQYFESPFETKYNCDTHCNKLCGK proteins BCR family, peptideBCR2 IDHCSCIQYHSMEGLWFPHCRTGSAAQMLHDFLSNP (SEQ ID NO: 66)Bacteriocyte-specific cysteine richMSVRKNVLPTMFVVLLIMSPVTPTSVFISAVCYSGCGSLA proteins BCR family, peptideBCR3 LVCFVSNGITNGLDYFKSSAPLSTSETSCGEAFDTCTDH CLANFKF (SEQ ID NO: 67)Bacteriocyte-specific cysteine richMRLLYGFLIIMLTIYLSVQDFDPTEFKGPFPTIEICSKYCAV proteins BCR family, peptideBCR4 VCNYTSRPCYCVEAAKERDQWFPYCYD (SEQ ID NO: 68) Bacteriocyte-specificcysteine rich MRLLYGFLIIMLTIHLSVQDIDPNTLRGPYPTKEICSKYCEY proteins BCRfamily, peptide BCR5 NVVCGASLPCICVQDARQLDHWFACCYDGGPEMLM (SEQ ID NO: 69)Secreted proteins SP family, peptideMKLFVVVVLVAVGIMFVFASDTAAAPTDYEDTNDMISLSS SP1LVGDNSPYVRVSSADSGGSSKTSSKNPILGLLKSVIKLLT KIFGTYSDAAPAMPPIPPALRKNRGMLA(SEQ ID NO: 70) Secreted proteins SP family, peptideMVACKVILAVAVVFVAAVQGRPGGEPEWAAPIFAELKSV SP2SDNITNLVGLDNAGEYATAAKNNLNAFAESLKTEAAVFSKSFEGKASASDVFKESTKNFQAVVDTYIKNLPKDLTLKDFTEKSEQALKYMVEHGTEITKKAQGNTETEKEIKEFFKKQIE NLIGQGKALQAKIAEAKKA (SEQ ID NO:71) Secreted proteins SP family, peptideMKTSSSKVFASCVAIVCLASVANALPVQKSVAATTENPIV SP3EKHGCRAHKNLVRQNVVDLKTYDSMLITNEVVQKQSNEVQSSEQSNEGQNSEQSNEGQNSEQSNEVQSSEHSNEGQNSKQSNEGQNSEQSNEVQSSEHSNEGQNSEQSNEVQSSEHSNEGQNSKQSNEGQNSKQSNEVQSSEHWNEGQNSKQSNEDQNSEQSNEGQNSKQSNEGQNSKQSNEDQNSEQSNEGQNSKQSNEVQSSEQSNEGQNSKQSNEGQSSEQSNEGQNSKQSNEVQSPEEHYDLPDPESSYESEETK GSHESGDDSEHR (SEQ ID NO: 72)Secreted proteins SP family, peptideMKTIILGLCLFGALFWSTQSMPVGEVAPAVPAVPSEAVP SP4QKQVEAKPETNAASPVSDAKPESDSKPVDAEVKPTVSEVKAESEQKPSGEPKPESDAKPVVASESKPESDPKPAAVVESKPENDAVAPETNNDAKPENAAAPVSENKPATDAKAETELIAQAKPESKPASDLKAEPEAAKPNSEVPVALPLNPTETKATQQSVETNQVEQAAPAAAQADPAAAPAADPAPAPAAAPVAAEEAKLSESAPSTENKAAEEPSKPAEQQSAKPVEDAVPAASEISETKVSPAVPAVPEVPASPSAPAVADPVSAPEAEKNAEPAKAANSAEPAVQSEAKPAEDIQKSGAVVSAENPKPVEEQKPAEVAKPAEQSKSEAPAEAPKPTEQSAAEEPKKPESANDEKKEQHSVNKRDATKEKKPTDSIMKKQKQKK AN (SEQ ID NO: 73) Secretedproteins SP family, peptide MNGKIVLCFAVVFIGQAMSAATGTTPEVEDIKKVAEQMS SP5aQTFMSVANHLVGITPNSADAQKSIEKIRTIMNKGFTDMETEANKMKDIVRKNADPKLVEKYDELEKELKKHLSTAKDMFEDKVVKPIGEKVELKKITENVIKTTKDMEATMNKAIDGFKKQ (SEQ ID NO: 74) Secretedproteins SP family, peptide MHLFLALGLFIVCGMVDATFYNPRSQTFNQLMERRQRSI SP6PIPYSYGYHYNPIEPSINVLDSLSEGLDSRINTFKPIYQNVKMSTQDVNSVPRTQYQPKNSLYDSEYISAKDIPSLFPEEDSYDYKYLGSPLNKYLTRPSTQESGIAINLVAIKETSVFDYGFPTYKSPYSSDSVWNFGSKIPNTVFEDPQSVESDPNTFKVSSPTIKIVKLLPETPEQESIITTTKNYELNYKTTQETPTEAELYPITSEEFQTEDEWHPMVPKENTTKDESSFITTEEPLTEDKSNSITIEKTQTEDESNSIEFNSIRTEEKSNSITTEENQKEDDESMSTTSQETTTAFNLNDTFDTNRYSSSHESLMLRIRELMKNIADQQNKSQFRTVDNIPAKSQSNLSSDESTNQ QFEPQLVNGADTYK (SEQ ID NO: 75)Coleoptericin A, ColA peptide MTRTMLFLACVAALYVCISATAGKPEEFAKLSDEAPSNDQAMYESIQRYRRFVDGNRYNGGQQQQQQPKQWEVRPDLSRDQRGNTKAQVEINKKGDNHDINAGWGKNINGPDS HKDTWHVGGSVRW (SEQ ID NO: 76)RlpA Type I MKETTVVWAKLFLILIILAKPLGLKAVNECKRLGNNSCRSHGECCSGFCFIEPGWALGVCKRLGTPKKSDDSNNGKNIEKNNGVHERIDDVFERGVCSYYKGPSITANGDVFDENEMTAAHRTLPFNTMVKVEGMGTSVVVKINDRKTAADGKVMLLSRAAAESLNIDENTGPVQCQLKFVLDGSGCTPDYGDTCVL HHECCSQNCFREMFSDKGFCLPK (SEQ IDNO: 77)

In some instances, the BRP alters the growth and/or activity of one ormore bacteria resident in the bacteriocyte of the host. In someinstances, the BRP may be bioengineered to modulate its bioactivity(e.g., increase, decrease, or regulate) or to specify a targetmicroorganism. In some instances, the BRP is produced by thetranslational machinery (e.g. a ribosome, etc.) of a cell. In someinstances, the BRP is chemically synthesized. In some instances, the BRPis derived from a polypeptide precursor. The polypeptide precursor canundergo cleavage (for example, processing by a protease) to yield thepolypeptide of the BRP itself. As such, in some instances, the BRP isproduced from a precursor polypeptide. In some instances, the BRPincludes a polypeptide that has undergone post-translationalmodifications, for example, cleavage, or the addition of one or morefunctional groups.

Functionally active variants of the BRPs as described herein are alsouseful in the compositions and methods described herein. In someinstances, the variant of the BRP has at least 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g.,over a specified region or over the entire sequence, to a sequence of aBRP described herein or naturally derived BRP.

The BRP described herein may be formulated in a composition for any ofthe uses described herein. The compositions disclosed herein may includeany number or type (e.g., classes) of BRPs, such as at least about anyone of 1 BRP, 2, 3, 4, 5, 10, 15, 20, or more BRPs. A suitableconcentration of each BRP in the composition depends on factors such asefficacy, stability of the BRP, number of distinct BRP, the formulation,and methods of application of the composition. In some instances, eachBRP in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL.In some instances, each BRP in a solid composition is from about 0.1ng/g to about 100 mg/g. In some instances, wherein the compositionincludes at least two types of BRPs, the concentration of each type ofBRP may be the same or different.

A modulating agent including a BRP as described herein can be contactedwith the target host in an amount and for a time sufficient to: (a)reach a target level (e.g., a predetermined or threshold level) of BRPconcentration inside a target host; (b) reach a target level (e.g., apredetermined or threshold level) of BRP concentration inside a targethost gut; (c) reach a target level (e.g., a predetermined or thresholdlevel) of BRP concentration inside a target host bacteriocyte; (d)modulate the level, or an activity, of one or more microorganism (e.g.,endosymbiont) in the target host; or/and (e) modulate fitness of thetarget host.

ii. Small Molecules

In some instances, the modulating agent includes a small molecule.Numerous small molecule agents are useful in the methods andcompositions described herein. The small molecules discussed hereinaftercan be used to alter pathways in host that mediate interactions betweenthe host and microorganisms resident in the host, as indicated in thesections for decreasing the fitness of insects, such as aphids.Additional small molecule agents can also be screened based on theirability to target components (e.g., polypeptides, e.g., enzymes or cellsurface receptors) of pathways in the host invertebrate (e.g., insect,mollusk, or nematode) and/or resident microorganism (e.g., polypeptidesthat mediate host-microbiota interactions, e.g., host immune systempathways or bacteriocyte pathways). In some instances, the smallmolecule includes an agonist, antagonist, inhibitor, or an activator.For example, a small molecule described herein may be an agonist,antagonist, inhibitor, or an activator that blocks or potentiatesactivity and/or function of a component of the host immune systempathway or bacteriocyte regulatory pathway listed in Table 8 or Table 9.The small molecule may act as an antagonist or agonist of a polypeptide(e.g., enzyme or cell receptor) in the host or microorganisms residentin the host, including any proteins list in Table 7, Table 8, or Table9.

Small molecules include, but are not limited to, small peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,synthetic polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic and inorganic compounds (includingheterorganic and organometallic compounds) generally having a molecularweight less than about 5,000 grams per mole, e.g., organic or inorganiccompounds having a molecular weight less than about 2,000 grams permole, e.g., organic or inorganic compounds having a molecular weightless than about 1,000 grams per mole, e.g., organic or inorganiccompounds having a molecular weight less than about 500 grams per mole,and salts, esters, and other pharmaceutically acceptable forms of suchcompounds.

The small molecule described herein may be formulated in a compositionfor any of the uses described herein. The compositions disclosed hereinmay include any number or type (e.g., classes) of small molecules, suchas at least about any one of 1 small molecule, 2, 3, 4, 5, 10, 15, 20,or more small molecules. A suitable concentration of each small moleculein the composition depends on factors such as efficacy, stability of thesmall molecule, number of distinct small molecules, the formulation, andmethods of application of the composition. In some instances, whereinthe composition includes at least two types of small molecules, theconcentration of each type of small molecule may be the same ordifferent.

A modulating agent including a small molecule as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of small molecule concentration inside a target host; (b) reach a targetlevel (e.g., a predetermined or threshold level) of small moleculeconcentration inside a target host gut; (c) reach a target level (e.g.,a predetermined or threshold level) of small molecule concentrationinside a target host bacteriocyte; (d) modulate the level, or anactivity, of one or more microorganism (e.g., endosymbiont) in thetarget host; or/and (e) modulate fitness of the target host.

In some instances, the small molecule triggers, stimulates, or increasesa host's immune response in comparison to a host organism to which thesmall molecule has not been administered. For example, the smallmolecule may be peptidoglycan molecule that activates the ROS system ininsects by binding to the epithelial cell surface, which in turnsinduces DUOX enzymatic activity by mobilizing intracellular calcium. Inanother example, the molecule dihydroxyphenylalanine (DOPA) is anessential component for cuticle synthesis. Once the cuticle is achieved,DOPA reaches high amounts in insects, which triggers apoptosis andautophagy activation. In another instance, the immune response iseffective to reduce the level of an endosymbiont or kill an endosymbiontin comparison to a host organism to which the small molecule has notbeen administered. In some instances, the small molecule is effective todisrupt or decrease bacteriocyte function in comparison to a hostorganism to which the modulating agent has not been administered. Forexample, molecules that block transport of essential amino acidprecursors inside the bacteriocyte also disrupt the production ofessential amino acids, e.g., arginine. This alteration ultimatelyresults in death of the endosymbiont, and, eventually, death of thehost. Other examples of modulating agents that can be used to stimulatea host's immune system and thereby reduce the levels of endosymbiontsresident in the host include lipopolysaccharides, rapamycin, andβ-glucan.

In some instances, the small molecule decreases or increases geneexpression of the resident microorganism by binding to non-coding RNAregion. For example, the small molecule may be a riboswitch inhibitor,such as ribocil, that binds to a ‘riboswitch’ regulatory domain in anon-coding region of the messenger RNA that encodes a synthase enzymeinvolved in riboflavin synthesis, therefore inhibiting this pathway. Inanother instance, the small molecule is effective to increase ordecrease gene expression that results in the killing of an endosymbiont.In some instances, the small molecule is effective to disruptbacteriocyte function.

In some instances, the small molecule alters a host's homeostasis. Forexample, the small molecule may be an eicosanoid molecule, such asprostaglandin, that activates a fever response to infection as well asin protein exocytosis in salivary glands. Aside from ongoing actions inhomeostasis, certain eicosanoid actions occur at crucial points ininsect life histories, such as during an infectious challenge andimportant events in reproduction. Eicosanoids mediate cellular defensereactions in insects. In one example, inhibition of prostaglandinsynthesis severely impairs the insects' ability to clear bacteria fromthe hemolymph. In another instance, the small molecule is effective toincrease or decrease an immune response in the host to an endosymbiontor increase or decrease a fever response in the host that results in thekilling of an endosymbiont. In some instances, the small molecule iseffective to disrupt bacteriocyte function.

As illustrated by Example 14, small molecules (e.g., prostaglandin) canbe used as modulating agents that target host pathways that, in turn,alter the activity, levels, or metabolism of endosymbiotic bacteria inthe host and thereby modulate (e.g., decrease) the fitness of the host.

iii. Nucleic Acids

Numerous nucleic acids are useful in the compositions and methodsdescribed herein. The compositions disclosed herein may include anynumber or type (e.g., classes) of nucleic acids (e.g., DNA molecule orRNA molecule, e.g., mRNA, guide RNA (gRNA), or inhibitory RNA molecule(e.g., siRNA, shRNA, or miRNA), or a hybrid DNA-RNA molecule), such asat least about 1 class or variant of a nucleic acid, 2, 3, 4, 5, 10, 15,20, or more classes or variants of nucleic acids. A suitableconcentration of each nucleic acid in the composition depends on factorssuch as efficacy, stability of the nucleic acid, number of distinctnucleic acids, the formulation, and methods of application of thecomposition. In some instances, wherein the composition includes atleast two types of nucleic acid, the concentration of each type ofnucleic acid may be the same or different.

A modulating agent including a nucleic acid as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of nucleic acid concentration inside a target host; (b) reach a targetlevel (e.g., a predetermined or threshold level) of nucleic acidconcentration inside a target host gut; (c) reach a target level (e.g.,a predetermined or threshold level) of nucleic acid concentration insidea target host bacteriocyte; (d) modulate the level, or an activity, ofone or more microorganism (e.g., endosymbiont) in the target host;or/and (e) modulate fitness of the target host.

The nucleic acid modulating agents discussed hereinafter, includingnucleic acids encoding polypeptides, synthetic RNA, inhibitory RNA, andgene editing systems, can be used to alter pathways in the host thatmediate interactions between the host and microorganisms resident in thehost as indicated in the sections for increasing or decreasing thefitness of hosts (e.g., aphids).

(a) Nucleic Acids Encoding a Polypeptide

In some instances, a composition includes a nucleic acid encoding anyone of the polypeptides described herein. Nucleic acids encoding apolypeptide may have a length from about 10 to about 50,000 nucleotides(nts), about 25 to about 100 nts, about 50 to about 150 nts, about 100to about 200 nts, about 150 to about 250 nts, about 200 to about 300nts, about 250 to about 350 nts, about 300 to about 500 nts, about 10 toabout 1000 nts, about 50 to about 1000 nts, about 100 to about 1000 nts,about 1000 to about 2000 nts, about 2000 to about 3000 nts, about 3000to about 4000 nts, about 4000 to about 5000 nts, about 5000 to about6000 nts, about 6000 to about 7000 nts, about 7000 to about 8000 nts,about 8000 to about 9000 nts, about 9000 to about 10,000 nts, about10,000 to about 15,000 nts, about 10,000 to about 20,000 nts, about10,000 to about 25,000 nts, about 10,000 to about 30,000 nts, about10,000 to about 40,000 nts, about 10,000 to about 45,000 nts, about10,000 to about 50,000 nts, or any range therebetween.

The modulating agent may also include functionally active variants ofthe nucleic acids described herein. In some instances, the variant ofthe nucleic acids has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over aspecified region or over the entire sequence, to a sequence of a nucleicacids described herein. In some instances, the invention includes afunctionally active polypeptide encoded by a nucleic acid variant asdescribed herein. In some instances, the functionally active polypeptideencoded by the nucleic acid variant has at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity,e.g., over a specified region or over the entire amino acid sequence, toa sequence of a polypeptide described herein or the naturally derivedpolypeptide sequence.

Some methods for expressing a nucleic acid encoding a protein mayinvolve expression in cells, including host cells (e.g., insect cells,mollusk cells, or nematode cells), yeast, bacteria, or other cells underthe control of appropriate promoters. Expression vectors may includenontranscribed elements, such as an origin of replication, a suitablepromoter and enhancer, and other 5′ or 3′ flanking nontranscribedsequences, and 5′ or 3′ nontranslated sequences such as necessaryribosome binding sites, a polyadenylation site, splice donor andacceptor sites, and termination sequences. DNA sequences derived fromthe SV40 viral genome, for example, SV40 origin, early promoter,enhancer, splice, and polyadenylation sites may be used to provide theother genetic elements required for expression of a heterologous DNAsequence. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described inGreen et al., Molecular Cloning: A Laboratory Manual, Fourth Edition,Cold Spring Harbor Laboratory Press, 2012.

Genetic modification using recombinant methods is generally known in theart. A nucleic acid sequence coding for a desired gene can be obtainedusing recombinant methods known in the art, such as, for example byscreening libraries from cells expressing the gene, by deriving the genefrom a vector known to include the same, or by isolating directly fromcells and tissues containing the same, using standard techniques.Alternatively, a gene of interest can be produced synthetically, ratherthan cloned.

Expression of natural or synthetic nucleic acids is typically achievedby operably linking a nucleic acid encoding the gene of interest to apromoter, and incorporating the construct into an expression vector.Expression vectors can be suitable for replication and expression inbacteria. Expression vectors can also be suitable for replication andintegration in eukaryotes. Typical cloning vectors contain transcriptionand translation terminators, initiation sequences, and promoters usefulfor expression of the desired nucleic acid sequence.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 basepairs (bp) upstream of the start site, although a number ofpromoters have recently been shown to contain functional elementsdownstream of the start site as well. The spacing between promoterelements frequently is flexible, so that promoter function is preservedwhen elements are inverted or moved relative to one another. In thethymidine kinase (tk) promoter, the spacing between promoter elementscan be increased to 50 bp apart before activity begins to decline.Depending on the promoter, it appears that individual elements canfunction either cooperatively or independently to activatetranscription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter.

Alternatively, the promoter may be an inducible promoter. The use of aninducible promoter provides a molecular switch capable of turning onexpression of the polynucleotide sequence which it is operatively linkedwhen such expression is desired, or turning off the expression whenexpression is not desired. Examples of inducible promoters include, butare not limited to a metallothionine promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline promoter.

The expression vector to be introduced can also contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes, such as neo and thelike.

Reporter genes may be used for identifying potentially transformed cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient source and that encodes a polypeptide whose expressionis manifested by some easily detectable property, e.g., enzymaticactivity. Expression of the reporter gene is assayed at a suitable timeafter the DNA has been introduced into the recipient cells. Suitablereporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., FEBS Letters 479:79-82, 2000). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In some instances, an organism may be genetically modified to alterexpression of one or more proteins. Expression of the one or moreproteins may be modified for a specific time, e.g., development ordifferentiation state of the organism. In one instances, the inventionincludes a composition to alter expression of one or more proteins,e.g., proteins that affect activity, structure, or function. Expressionof the one or more proteins may be restricted to a specific location(s)or widespread throughout the organism.

(b) Synthetic mRNA

The modulating agent may include an mRNA molecule, e.g., a syntheticmRNA molecule encoding a polypeptide. In some instances, the mRNAmolecule increases the level (e.g., protein and/or mRNA level) and/oractivity of an agent, e.g., a positive regulator of function, e.g., agene or gene product listed in Table 7, Table 8, or Table 9. In someinstances, the mRNA molecule encodes a polypeptide agent or a fragmentthereof. For example, the mRNA molecule may encode a polypeptide havingat least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, orgreater) identity to the amino acid sequence of an agent listed in Table7, Table 8, or Table 9 all with reference to accession number provided.In other examples, the mRNA molecule has at least 50% (e.g., at least50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to thenucleic acid sequence encoding an agent listed in Table 7, Table 8, orTable 9. In some instances, the mRNA molecule encodes an amino acidsequence differing by no more than 30 (e.g., no more than 30, 20, 10, 5,4, 3, 2, or 1) amino acids to the amino acid sequence of an agent listedin Table 7, Table 8, or Table 9 all with reference to accession numberprovided. In some instances, the mRNA molecule includes a sequenceencoding a fragment of a gene or gene product listed in Table 7, Table8, or Table 9 all with reference to accession number provided. Forexample, the fragment includes 10-20, 20-40, 40-60, 60-80, 80-100,100-120, 120-140, 140-160, 160-180, 180-200, 200-250, 250-300, 300-400,400-500, 500-600, or more amino acids in length. In some instances, thefragment is a functional fragment, e.g., having at least 20%, e.g., atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater, of an activityof a full length gene or gene product listed in Table 7, Table 8, orTable 9 all with reference to accession numbers provided. In someinstances, the mRNA molecule increases the level and/or activity of orencodes an agent (or fragment thereof).

An exemplary mRNA molecule includes an RNA encoding any polypeptideselected from Table 7, Table 8, or Table 9.

The synthetic mRNA molecule can be modified, e.g., chemically. The mRNAmolecule can be chemically synthesized or transcribed in vitro. The mRNAmolecule can be disposed on a plasmid, e.g., a viral vector, bacterialvector, or eukaryotic expression vector. In some examples, the mRNAmolecule can be delivered to cells by transfection, electroporation, ortransduction (e.g., adenoviral or lentiviral transduction).

In some instances, the modified RNA agent of interest described hereinhas modified nucleosides or nucleotides. Such modifications are knownand are described, e.g., in WO 2012/019168. Additional modifications aredescribed, e.g., in WO 2015/038892; WO 2015/038892; WO 2015/089511; WO2015/196130; WO 2015/196118 and WO 201 5/1 961 28 A2.

In some instances, the modified RNA encoding a polypeptide of interestdescribed herein has one or more terminal modification, e.g., a 5′ capstructure and/or a poly-A tail (e.g., of between 100-200 nucleotides inlength). The 5′ cap structure may be selected from the group consistingof CapO, Capl, ARCA, inosine, NI-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine. In some cases, the modified RNAs also contain a 5‘ UTR including at least one Kozak sequence, and a 3’ UTR. Suchmodifications are known and are described, e.g., in WO 2012/135805 andWO 2013/052523. Additional terminal modifications are described, e.g.,in WO 2014/164253 and WO 2016/011306, WO 2012/045075, and WO2014/093924.

Chimeric enzymes for synthesizing capped RNA molecules (e.g., modifiedmRNA) which may include at least one chemical modification are describedin WO 2014/028429.

In some instances, a modified mRNA may be cyclized, or concatemerized,to generate a translation competent molecule to assist interactionsbetween poly-A binding proteins and 5′-end binding proteins. Themechanism of cyclization or concatemerization may occur through at least3 different routes: 1) chemical, 2) enzymatic, and 3) ribozymecatalyzed. The newly formed 5′-/3′-linkage may be intramolecular orintermolecular. Such modifications are described, e.g., in WO2013/151736.

Methods of making and purifying modified RNAs are known and disclosed inthe art. For example, modified RNAs are made using only in vitrotranscription (IVT) enzymatic synthesis. Methods of making IVTpolynucleotides are known in the art and are described in WO2013/151666, WO 2013/151668, WO 2013/151663, WO 2013/151669, WO2013/151670, WO 2013/151664, WO 2013/151665, WO 2013/151671, WO2013/151672, WO 201 3/1 51 667 and WO 2013/151736.S Methods ofpurification include purifying an RNA transcript including a polyA tailby contacting the sample with a surface linked to a plurality ofthymidines or derivatives thereof and/or a plurality of uracils orderivatives thereof (polyT/U) under conditions such that the RNAtranscript binds to the surface and eluting the purified RNA transcriptfrom the surface (WO 2014/152031); using ion (e.g., anion) exchangechromatography that allows for separation of longer RNAs up to 10,000nucleotides in length via a scalable method (WO 2014/144767); andsubjecting a modified mRNA sample to DNAse treatment (WO 2014/152030).

Formulations of modified RNAs are known and are described, e.g., in WO2013/090648. For example, the formulation may be, but is not limited to,nanoparticles, poly(lactic-co-glycolic acid)(PLGA) microspheres,lipidoids, lipoplex, liposome, polymers, carbohydrates (including simplesugars), cationic lipids, fibrin gel, fibrin hydrogel, fibrin glue,fibrin sealant, fibrinogen, thrombin, rapidly eliminated lipidnanoparticles (reLNPs) and combinations thereof.

Modified RNAs encoding polypeptides in the fields of human disease,antibodies, viruses, and a variety of in vivo settings are known and aredisclosed in for example, Table 6 of International Publication Nos. WO2013/151666, WO 2013/151668, WO 2013/151663, WO 2013/151669, WO2013/151670, WO 2013/151664, WO 2013/151665, WO 2013/151736; Tables 6and 7 International Publication No. WO 2013/151672; Tables 6, 178 and179 of International Publication No. WO 2013/151671; Tables 6, 185 and186 of International Publication No WO 2013/151667. Any of the foregoingmay be synthesized as an IVT polynucleotide, chimeric polynucleotide ora circular polynucleotide, and each may include one or more modifiednucleotides or terminal modifications.

(c) Inhibitory RNA

In some instances, the modulating agent includes an inhibitory RNAmolecule, e.g., that acts via the RNA interference (RNAi) pathway. Forexample, an inhibitory RNA molecule may include a short interfering RNA,short hairpin RNA, and/or a microRNA that targets host pathways (e.g.,pathways that mediate host-microbiota interactions, e.g., host immunesystem pathways or bacteriocyte pathways, e.g., proteins or genesencoding proteins listed in Table 8 or Table 9, all with reference toaccession number provided) in the host invertebrate (e.g., insect,mollusk, or nematode) and/or pathways in the resident microorganisms(e.g., proteins or genes encoding proteins listed in Table 7, all withreference to accession number provided). Certain RNA molecules caninhibit gene expression through the biological process of RNAinterference (RNAi). RNAi molecules include RNA or RNA-like structurestypically containing 15-50 base pairs (such as about 18-25 base pairs)and having a nucleobase sequence identical (complementary) or nearlyidentical (substantially complementary) to a coding sequence in anexpressed target gene within the cell. RNAi molecules include, but arenot limited to: short interfering RNAs (siRNAs), double-strand RNAs(dsRNA), short hairpin RNAs (shRNA), meroduplexes, dicer substrates, andmultivalent RNA interference (U.S. Pat. Nos. 8,084,599 8,349,809,8,513,207 and 9,200,276). A shRNA is a RNA molecule comprising a hairpinturn that decreases expression of target genes via RNAi. shRNAs can bedelivered to cells in the form of plasmids, e.g., viral or bacterialvectors, e.g., by transfection, electroporation, or transduction). AmicroRNA is a non-coding RNA molecule that typically has a length ofabout 22 nucleotides. MiRNAs bind to target sites on mRNA molecules andsilence the mRNA, e.g., by causing cleavage of the mRNA, destabilizationof the mRNA, or inhibition of translation of the mRNA. In someinstances, the inhibitory RNA molecule decreases the level and/oractivity of a negative regulator of function. In other embodiments, theinhibitor RNA molecule decreases the level and/or activity of aninhibitor of a positive regulator of function.

RNAi molecules include a sequence substantially complementary, or fullycomplementary, to all or a fragment of a target gene. RNAi molecules maycomplement sequences at the boundary between introns and exons toprevent the maturation of newly-generated nuclear RNA transcripts ofspecific genes into mRNA for transcription. RNAi molecules complementaryto specific genes can hybridize with the mRNA for a target gene andprevent its translation. The antisense molecule can be DNA, RNA, or aderivative or hybrid thereof. Examples of such derivative moleculesinclude, but are not limited to, peptide nucleic acid (PNA) andphosphorothioate-based molecules such as deoxyribonucleic guanidine(DNG) or ribonucleic guanidine (RNG).

RNAi molecules can be provided as “ready-to-use” RNA synthesized invitro or as an antisense gene transfected into cells which will yieldRNAi molecules upon transcription. Hybridization with mRNA results indegradation of the hybridized molecule by RNAse H and/or inhibition ofthe formation of translation complexes. Both result in a failure toproduce the product of the original gene.

The length of the RNAi molecule that hybridizes to the transcript ofinterest may be around 10 nucleotides, between about 15 or 30nucleotides, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more nucleotides. The degree of identity of theantisense sequence to the targeted transcript may be at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95.

RNAi molecules may also include overhangs, i.e., typically unpaired,overhanging nucleotides which are not directly involved in the doublehelical structure normally formed by the core sequences of the hereindefined pair of sense strand and antisense strand. RNAi molecules maycontain 3′ and/or 5′ overhangs of about 1-5 bases independently on eachof the sense strands and antisense strands. In some instances, both thesense strand and the antisense strand contain 3′ and 5′ overhangs. Insome instances, one or more of the 3′ overhang nucleotides of one strandbase pairs with one or more 5′ overhang nucleotides of the other strand.In other instances, the one or more of the 3′ overhang nucleotides ofone strand base do not pair with the one or more 5′ overhang nucleotidesof the other strand. The sense and antisense strands of an RNAi moleculemay or may not contain the same number of nucleotide bases. Theantisense and sense strands may form a duplex wherein the 5′ end onlyhas a blunt end, the 3′ end only has a blunt end, both the 5′ and 3′ends are blunt ended, or neither the 5′ end nor the 3′ end are bluntended. In another instance, one or more of the nucleotides in theoverhang contains a thiophosphate, phosphorothioate, deoxynucleotideinverted (3′ to 3′ linked) nucleotide or is a modified ribonucleotide ordeoxynucleotide.

Small interfering RNA (siRNA) molecules include a nucleotide sequencethat is identical to about 15 to about 25 contiguous nucleotides of thetarget mRNA. In some instances, the siRNA sequence commences with thedinucleotide AA, includes a GC-content of about 30-70% (about 30-60%,about 40-60%, or about 45%-55%), and does not have a high percentageidentity to any nucleotide sequence other than the target in the genomein which it is to be introduced, for example as determined by standardBLAST search.

siRNAs and shRNAs resemble intermediates in the processing pathway ofthe endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004).In some instances, siRNAs can function as miRNAs and vice versa (Zeng etal., Mol. Cell 9:1327-1333, 2002; Doench et al., Genes Dev. 17:438-442,2003). Exogenous siRNAs downregulate mRNAs with seed complementarity tothe siRNA (Birmingham et al., Nat. Methods 3:199-204, 2006). Multipletarget sites within a 3′ UTR give stronger downregulation (Doench etal., Genes Dev. 17:438-442, 2003).

Known effective siRNA sequences and cognate binding sites are also wellrepresented in the relevant literature. RNAi molecules are readilydesigned and produced by technologies known in the art. In addition,there are computational tools that increase the chance of findingeffective and specific sequence motifs (Pei et al., Nat. Methods3(9):670-676, 2006; Reynolds et al., Nat. Biotechnol. 22(3):326-330,2004; Khvorova et al., Nat. Struct. Biol. 10(9):708-712, 2003; Schwarzet al., Cell 115(2):199-208, 2003; Ui-Tei et al., Nucleic Acids Res.32(3):936-948, 2004; Heale et al., Nucleic Acids Res. 33(3):e30, 2005;Chalk et al., Biochem. Biophys. Res. Commun. 319(1):264-274, 2004; andAmarzguioui et al., Biochem. Biophys. Res. Commun. 316(4):1050-1058,2004).

The RNAi molecule modulates expression of RNA encoded by a gene. Becausemultiple genes can share some degree of sequence homology with eachother, in some instances, the RNAi molecule can be designed to target aclass of genes with sufficient sequence homology. In some instances, theRNAi molecule can contain a sequence that has complementarity tosequences that are shared amongst different gene targets or are uniquefor a specific gene target. In some instances, the RNAi molecule can bedesigned to target conserved regions of an RNA sequence having homologybetween several genes thereby targeting several genes in a gene family(e.g., different gene isoforms, splice variants, mutant genes, etc.). Insome instances, the RNAi molecule can be designed to target a sequencethat is unique to a specific RNA sequence of a single gene.

In some instances, the inhibitory RNA molecule decreases the leveland/or activity of a host component (e.g., component in pathways thatmediate host-microbiota interactions, e.g., host immune system pathwaysor bacteriocyte pathways) and/or microbial component, including genesencoding proteins listed in Table 7, Table 8, or Table 9, all withreference to accession number provided. In some instances, theinhibitory RNA molecule inhibits expression of a host component (e.g.,component in pathways that mediate host-microbiota interactions, e.g.,host immune system pathways or bacteriocyte pathways) or microbialcomponent, e.g., genes encoding proteins listed in Table 7, Table 8, orTable 9 (e.g., inhibits translation to protein). In other instances, theinhibitor RNA molecule increases degradation of a host component (e.g.,component in pathways that mediate host-microbiota interactions, e.g.,host immune system pathways or bacteriocyte pathways) and/or microbialcomponent, e.g., genes encoding proteins listed in Table 7, Table 8, orTable 9 and/or decreases the stability (i.e., half-life) of the pathwaycomponent. The inhibitory RNA molecule can be chemically synthesized ortranscribed in vitro.

In some instances, the compositions described herein include an RNAi,e.g., siRNA, to regulate (e.g., inhibit) expression of a gene encodingany of the components described herein that regulate a host's immunesystem. In some instances, a composition includes an RNAi, e.g., siRNA,to inhibit expression of any one of the genes described herein thatregulate (e.g., inhibit) the development or function of a bacteriocytein the host. In some instances, regulation of the host immune systemleads to a reduction or killing of an endosymbiotic microorganism in thehost, and in turn, reduces the fitness of the host. In some instances,regulation of bacteriocyte development and/or function leads to areduction or killing of an endosymbiotic microorganism in the host, andin turn, reduces the fitness of the host. In some instances, the RNAi(e.g., siRNA) may inhibit expression of Ubx to disrupt symbiontlocalization of bacteriocytes. In some instances, the RNAi (e.g., siRNA)inhibits expression of abd-A and Antp to disrupt bacteriome integrityand positioning.

In some instances, one or more RNAi molecules target any gene encodingany protein described herein, e.g., see Table 7, Table 8, or Table 9. Insome instances, one or more RNAi molecules target a bacterial gene asdescribed herein. In some instances, one or more RNAi molecules targetan endosymbiont gene as described herein. In some instances, one or moreRNAi molecules target a bacteriocyte gene as described herein. In someinstances, one or more RNAi molecules target a host gene as describedherein. In some instances, one or more RNAi molecules target an immunesystem gene in a host as described herein.

An inhibitory RNA molecule can be modified, e.g., to contain modifiednucleotides, e.g., 2′-fluoro, 2′-o-methyl, 2′-deoxy, unlocked nucleicacid, 2′-hydroxy, phosphorothioate, 2′-thiouridine, 4′-thiouridine,2′-deoxyuridine. Without being bound by theory, it is believed that suchmodifications can increase nuclease resistance and/or serum stability,or decrease immunogenicity.

In some instances, the RNAi molecule is linked to a delivery polymer viaa physiologically labile bond or linker. The physiologically labilelinker is selected such that it undergoes a chemical transformation(e.g., cleavage) when present in certain physiological conditions,(e.g., disulfide bond cleaved in the reducing environment of the cellcytoplasm). Release of the molecule from the polymer, by cleavage of thephysiologically labile linkage, facilitates interaction of the moleculewith the appropriate cellular components for activity.

The RNAi molecule-polymer conjugate may be formed by covalently linkingthe molecule to the polymer. The polymer is polymerized or modified suchthat it contains a reactive group A. The RNAi molecule is alsopolymerized or modified such that it contains a reactive group B.Reactive groups A and B are chosen such that they can be linked via areversible covalent linkage using methods known in the art.

Conjugation of the RNAi molecule to the polymer can be performed in thepresence of an excess of polymer. Because the RNAi molecule and thepolymer may be of opposite charge during conjugation, the presence ofexcess polymer can reduce or eliminate aggregation of the conjugate.Alternatively, an excess of a carrier polymer, such as a polycation, canbe used. The excess polymer can be removed from the conjugated polymerprior to administration of the conjugate. Alternatively, the excesspolymer can be co-administered with the conjugate.

Injection of double-stranded RNA (dsRNA) into mother insects efficientlysuppresses their offspring's gene expression during embryogenesis, seefor example, Khila et al., PLoS Genet. 5(7):e1000583, 2009; and Liu etal., Development 131(7):1515-1527, 2004. Matsuura et al. (PNAS112(30):9376-9381, 2015) has shown that suppression of Ubx eliminatesbacteriocytes and the symbiont localization of bacteriocytes.

The making and use of inhibitory agents based on non-coding RNA such asribozymes, RNAse P, siRNAs, and miRNAs are also known in the art, forexample, as described in Sioud, RNA Therapeutics: Function, Design, andDelivery (Methods in Molecular Biology). Humana Press (2010).

Other examples of nucleic acid modulating agents that can be used hereininclude dsRNAs having at least 50% (e.g., at least 50%, 60%, 70%, 80%,90%, 95%, 97%, 99%, or greater) identity to the sequence of any one ofSEQ ID NOs: 148-150.

As illustrated by Examples 1-4 and 7-9, inhibitory RNA (e.g., dsRNA orPNA) can be used as a modulating agent that targets a host pathway(e.g., an insect, e.g., an aphid) that, in turn, alters the activity,levels, or metabolism of endosymbiotic bacteria, such as a Buchneraspp., resident in the host and thereby modulates (e.g., decreases) thefitness of the host.

(d) Gene Editing

The modulating agents described herein may include a component of a geneediting system. For example, the agent may introduce an alteration(e.g., insertion, deletion (e.g., knockout), translocation, inversion,single point mutation, or other mutation) in a gene related to theimmune system or bacteriocyte of a host invertebrate (e.g., insect,mollusk, or nematode) or in a gene in a microorganism resident in thehost invertebrate, e.g., an enzyme or receptor gene described in Table7, Table 8, or Table 9 all with reference to accession number provided.Exemplary gene editing systems include the zinc finger nucleases (ZFNs),Transcription Activator-Like Effector-based Nucleases (TALEN), and theclustered regulatory interspaced short palindromic repeat (CRISPR)system. ZFNs, TALENs, and CRISPR-based methods are described, e.g., inGaj et al., Trends Biotechnol. 31(7):397-405, 2013.

In a typical CRISPR/Cas system, an endonuclease is directed to a targetnucleotide sequence (e.g., a site in the genome that is to besequence-edited) by sequence-specific, non-coding “guide RNAs” thattarget single- or double-stranded DNA sequences. Three classes (I-III)of CRISPR systems have been identified. The class II CRISPR systems usea single Cas endonuclease (rather than multiple Cas proteins). One classII CRISPR system includes a type II Cas endonuclease such as Cas9, aCRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). ThecrRNA contains a “guide RNA,” i.e., typically an about 20-nucleotide RNAsequence that corresponds to a target DNA sequence. The crRNA alsocontains a region that binds to the tracrRNA to form a partiallydouble-stranded structure which is cleaved by RNase III, resulting in acrRNA/tracrRNA hybrid. The RNAs serve as guides to direct Cas proteinsto silence specific DNA/RNA sequences, depending on the spacer sequence.See, e.g., Horvath et al., Science 327:167-170, 2010; Makarova et al.,Biology Direct 1:7, 2006; Pennisi, Science 341:833-836, 2013. The targetDNA sequence must generally be adjacent to a “protospacer adjacentmotif” (“PAM”) that is specific for a given Cas endonuclease; however,PAM sequences appear throughout a given genome. CRISPR endonucleasesidentified from various prokaryotic species have unique PAM sequencerequirements; examples of PAM sequences include 5′-NGG (SEQ ID NO: 78)(Streptococcus pyogenes), 5′-NNAGAA (SEQ ID NO: 79) (Streptococcusthermophilus CRISPR1), 5′-NGGNG (SEQ ID NO: 80) (Streptococcusthermophilus CRISPR3), and 5′-NNNGATT (SEQ ID NO: 81) (Neisseriameningiditis). Some endonucleases, e.g., Cas9 endonucleases, areassociated with G-rich PAM sites, e.g., 5′-NGG (SEQ ID NO: 78), andperform blunt-end cleaving of the target DNA at a location 3 nucleotidesupstream from (5′ from) the PAM site. Another class II CRISPR systemincludes the type V endonuclease Cpf1, which is smaller than Cas9;examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (fromLachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed intomature crRNAs without the requirement of a tracrRNA; in other words aCpf1 system requires only the Cpf1 nuclease and a crRNA to cleave thetarget DNA sequence. Cpf1 endonucleases, are associated with T-rich PAMsites, e.g., 5′-TTN. Cpf1 can also recognize a 5′-CTA PAM motif. Cpf1cleaves the target DNA by introducing an offset or staggereddouble-strand break with a 4- or 5-nucleotide 5′ overhang, for example,cleaving a target DNA with a 5-nucleotide offset or staggered cutlocated 18 nucleotides downstream from (3′ from) from the PAM site onthe coding strand and 23 nucleotides downstream from the PAM site on thecomplimentary strand; the 5-nucleotide overhang that results from suchoffset cleavage allows more precise genome editing by DNA insertion byhomologous recombination than by insertion at blunt-end cleaved DNA.See, e.g., Zetsche et al., Cell 163:759-771, 2015.

For the purposes of gene editing, CRISPR arrays can be designed tocontain one or multiple guide RNA sequences corresponding to a desiredtarget DNA sequence; see, for example, Cong et al., Science 339:819-823,2013; Ran et al., Nature Protocols 8:2281-2308, 2013. At least about 16or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavageto occur; for Cpf1 at least about 16 nucleotides of gRNA sequence isneeded to achieve detectable DNA cleavage. In practice, guide RNAsequences are generally designed to have a length of between 17-24nucleotides (e.g., 19, 20, or 21 nucleotides) and complementarity to thetargeted gene or nucleic acid sequence. Custom gRNA generators andalgorithms are available commercially for use in the design of effectiveguide RNAs. Gene editing has also been achieved using a chimeric “singleguide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule thatmimics a naturally occurring crRNA-tracrRNA complex and contains both atracrRNA (for binding the nuclease) and at least one crRNA (to guide thenuclease to the sequence targeted for editing). Chemically modifiedsgRNAs have also been demonstrated to be effective in genome editing;see, for example, Hendel et al., Nature Biotechnol. 985-991, 2015.

Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specificDNA sequences targeted by a gRNA, a number of CRISPR endonucleaseshaving modified functionalities are available, for example: a “nickase”version of Cas9 generates only a single-strand break; a catalyticallyinactive Cas9 (“dCas9”) does not cut the target DNA but interferes withtranscription by steric hindrance. dCas9 can further be fused with aneffector to repress (CRISPRi) or activate (CRISPRa) expression of atarget gene. For example, Cas9 can be fused to a transcriptionalrepressor (e.g., a KRAB domain) or a transcriptional activator (e.g., adCas9-VP64 fusion). A catalytically inactive Cas9 (dCas9) fused to FokInuclease (“dCas9-FokI”) can be used to generate DSBs at target sequenceshomologous to two gRNAs. See, e.g., the numerous CRISPR/Cas9 plasmidsdisclosed in and publicly available from the Addgene repository(Addgene, 75 Sidney St., Suite 550A, Cambridge, Mass. 02139;addgene.org/crispr/). A “double nickase” Cas9 that introduces twoseparate double-strand breaks, each directed by a separate guide RNA, isdescribed as achieving more accurate genome editing by Ran et al., Cell154:1380-1389, 2013.

CRISPR technology for editing the genes of eukaryotes is disclosed in USPatent Application Publications US 2016/0138008 A1 and US 2015/0344912A1, and in U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641,8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356,8,932,814, 8,795,965, and 8,906,616. Cpf1 endonuclease and correspondingguide RNAs and PAM sites are disclosed in US Patent ApplicationPublication 2016/0208243 A1.

In some instances, the desired genome modification involves homologousrecombination, wherein one or more double-stranded DNA breaks in thetarget nucleotide sequence is generated by the RNA-guided nuclease andguide RNA(s), followed by repair of the break(s) using a homologousrecombination mechanism (“homology-directed repair”). In such instances,a donor template that encodes the desired nucleotide sequence to beinserted or knocked-in at the double-stranded break is provided to thecell or subject; examples of suitable templates include single-strandedDNA templates and double-stranded DNA templates (e.g., linked to thepolypeptide described herein). In general, a donor template encoding anucleotide change over a region of less than about 50 nucleotides isprovided in the form of single-stranded DNA; larger donor templates(e.g., more than 100 nucleotides) are often provided as double-strandedDNA plasmids. In some instances, the donor template is provided to thecell or subject in a quantity that is sufficient to achieve the desiredhomology-directed repair but that does not persist in the cell orsubject after a given period of time (e.g., after one or more celldivision cycles). In some instances, a donor template has a corenucleotide sequence that differs from the target nucleotide sequence(e.g., a homologous endogenous genomic region) by at least 1, at least5, at least 10, at least 20, at least 30, at least 40, at least 50, ormore nucleotides. This core sequence is flanked by “homology arms” orregions of high sequence identity with the targeted nucleotide sequence;in some instances, the regions of high identity include at least 10, atleast 50, at least 100, at least 150, at least 200, at least 300, atleast 400, at least 500, at least 600, at least 750, or at least 1000nucleotides on each side of the core sequence. In some instances wherethe donor template is in the form of a single-stranded DNA, the coresequence is flanked by homology arms including at least 10, at least 20,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, or at least 100 nucleotides on each side of the core sequence.In instances, where the donor template is in the form of adouble-stranded DNA, the core sequence is flanked by homology armsincluding at least 500, at least 600, at least 700, at least 800, atleast 900, or at least 1000 nucleotides on each side of the coresequence. In one instance, two separate double-strand breaks areintroduced into the cell or subject's target nucleotide sequence with a“double nickase” Cas9 (see Ran et al., Cell 154:1380-1389, 2013),followed by delivery of the donor template.

In some instances, the composition includes a gRNA and a targetednuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9 (e.g.,Cas9 D10A), a dead Cas9 (dCas9), eSpCas9, Cpf1, C2C1, or C2C3, or anucleic acid encoding such a nuclease. The choice of nuclease andgRNA(s) is determined by whether the targeted mutation is a deletion,substitution, or addition of nucleotides, e.g., a deletion,substitution, or addition of nucleotides to a targeted sequence. Fusionsof a catalytically inactive endonuclease e.g., a dead Cas9 (dCas9, e.g.,D10A; H840A) tethered with all or a portion of (e.g., biologicallyactive portion of) an (one or more) effector domain create chimericproteins that can be linked to the polypeptide to guide the compositionto specific DNA sites by one or more RNA sequences (sgRNA) to modulateactivity and/or expression of one or more target nucleic acidssequences.

In instances, the agent includes a guide RNA (gRNA) for use in a CRISPRsystem for gene editing. In some instances, the agent includes a zincfinger nuclease (ZFN), or a mRNA encoding a ZFN, that targets (e.g.,cleaves) a nucleic acid sequence (e.g., DNA sequence) of a gene relatedto pathways in the host invertebrate (e.g., insect, mollusk, ornematode) and/or resident microorganisms (e.g., pathways that mediatehost-microbiota interactions, e.g., host immune system pathways orbacteriocyte pathways, e.g., a gene encoding a protein listed in Table7, Table 8, or Table 9, all with reference to accession numberprovided). In some instances, the agent includes a TALEN, or an mRNAencoding a TALEN, that targets (e.g., cleaves) a nucleic acid sequence(e.g., DNA sequence) in a gene related to pathways in the hostinvertebrate (e.g., insect, mollusk, or nematode) and/or residentmicroorganisms (e.g., pathways that mediate host-microbiotainteractions, e.g., host immune system pathways or bacteriocytepathways, e.g., a gene encoding a protein listed in Table 7, Table 8, orTable 9, all with reference to accession number provided).

For example, the gRNA can be used in a CRISPR system to engineer analteration in a gene related to pathways in the host invertebrate (e.g.,insect, mollusk, or nematode) and/or resident microorganisms (e.g.,pathways that mediate host-microbiota interactions, e.g., host immunesystem pathways or bacteriocyte pathways, e.g., a gene encoding aprotein listed in Table 7, Table 8, or Table 9, all with reference toaccession number provided). In other examples, the ZFN and/or TALEN canbe used to engineer an alteration in a gene related to pathways in thehost invertebrate (e.g., insect, mollusk, or nematode) and/or residentmicroorganisms (e.g., pathways that mediate host-microbiotainteractions, e.g., host immune system pathways or bacteriocytepathways, e.g., a gene encoding a protein listed in Table 7, Table 8, orTable 9, all with reference to accession number provided). Exemplaryalterations include insertions, deletions (e.g., knockouts),translocations, inversions, single point mutations, or other mutations.The alteration can be introduced in the gene in a cell, e.g., in vitro,ex vivo, or in vivo. In some examples, the alteration increases thelevel and/or activity of a gene related to pathways in the hostinvertebrate (e.g., insect, mollusk, or nematode) and/or residentmicroorganisms (e.g., pathways that mediate host-microbiotainteractions, e.g., host immune system pathways or bacteriocytepathways, e.g., a gene encoding a protein listed in Table 7, Table 8, orTable 9, all with reference to accession number provided). In otherexamples, the alteration decreases the level and/or activity of (e.g.,knocks down or knocks out) a gene related to pathways in the hostinvertebrate (e.g., insect, mollusk, or nematode) and/or residentmicroorganisms (e.g., pathways that mediate host-microbiotainteractions, e.g., host immune system pathways or bacteriocytepathways, e.g., a gene encoding a protein listed in Table 7, Table 8, orTable 9, all with reference to accession number provided). In yetanother example, the alteration corrects a defect (e.g., a mutationcausing a defect), in a gene related to pathways in the hostinvertebrate (e.g., insect, mollusk, or nematode) and/or residentmicroorganisms (e.g., pathways that mediate host-microbiotainteractions, e.g., host immune system pathways or bacteriocytepathways, e.g., a gene encoding protein listed in Table 7, Table 8, orTable 9, all with reference to accession number provided).

In some instances, the CRISPR system is used to edit (e.g., to add ordelete a base pair) a target gene (e.g., a gene related to pathways inthe host invertebrate (e.g., insect, mollusk, or nematode) and/orresident microorganisms, e.g., pathways that mediate host-microbiotainteractions, e.g., host immune system pathways or bacteriocytepathways, e.g., a gene encoding a protein listed in Table 7, Table 8, orTable 9, all with reference to accession number provided). In otherinstances, the CRISPR system is used to introduce a premature stopcodon, e.g., thereby decreasing the expression of a target gene. In yetother instances, the CRISPR system is used to turn off a target gene ina reversible manner, e.g., similarly to RNA interference. In someinstances, the CRISPR system is used to direct Cas to a promoter of agene, thereby blocking an RNA polymerase sterically.

In some instances, a CRISPR system can be generated to edit a generelated to pathways in the host invertebrate (e.g., insect, mollusk, ornematode) and/or resident microorganisms (e.g., pathways that mediatehost-microbiota interactions, e.g., host immune system pathways orbacteriocyte pathways, e.g., genes that encode a protein listed in Table7, Table 8, or Table 9, all with reference to accession numberprovided), using technology described in, e.g., U.S. Publication No.20140068797, Cong, Science 339: 819-823, 2013; Tsai, Nature Biotechnol.32:6 569-576, 2014; U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965;8,771,945; and 8,697,359.

In some instances, the CRISPR interference (CRISPRi) technique can beused for transcriptional repression of specific genes, e.g., a geneencoding a host immune system or bacteriocyte component (e.g., an enzymeor receptor described herein). In CRISPRi, an engineered Cas9 protein(e.g., nuclease-null dCas9, or dCas9 fusion protein, e.g., dCas9-KRAB ordCas9-SID4X fusion) can pair with a sequence specific guide RNA (sgRNA).The Cas9-gRNA complex can block RNA polymerase, thereby interfering withtranscription elongation. The complex can also block transcriptioninitiation by interfering with transcription factor binding. The CRISPRimethod is specific with minimal off-target effects and is multiplexable,e.g., can simultaneously repress more than one gene (e.g., usingmultiple gRNAs). Also, the CRISPRi method permits reversible generepression.

In some instances, CRISPR-mediated gene activation (CRISPRa) can be usedfor transcriptional activation, e.g., of one or more genes describedherein (e.g., a gene encoding any of the proteins listed in Table 7,Table 8, or Table 9). In the CRISPRa technique, dCas9 fusion proteinsrecruit transcriptional activators. For example, dCas9 can be fused topolypeptides (e.g., activation domains) such as VP64 or the p65activation domain (p65D) and used with sgRNA (e.g., a single sgRNA ormultiple sgRNAs), to activate a gene or genes, e.g., endogenous gene(s)in the host or microorganism resident in the host. Multiple activatorscan be recruited by using multiple sgRNAs—this can increase activationefficiency. A variety of activation domains and single or multipleactivation domains can be used. In addition to engineering dCas9 torecruit activators, sgRNAs can also be engineered to recruit activators.For example, RNA aptamers can be incorporated into a sgRNA to recruitproteins (e.g., activation domains) such as VP64. In some examples, thesynergistic activation mediator (SAM) system can be used fortranscriptional activation. In SAM, MS2 aptamers are added to the sgRNA.MS2 recruits the MS2 coat protein (MCP) fused to p65AD and heat shockfactor 1 (HSF1).

The CRISPRi and CRISPRa techniques are described in greater detail,e.g., in Dominguez et al., Nat. Rev. Mol. Cell Biol. 17:5-15, 2016,incorporated herein by reference. In addition, dCas9-mediated epigeneticmodifications and simultaneous activation and repression using CRISPRsystems, as described in Dominguez et al., can be used to modulate acomponent of a host or microbial pathway described herein (e.g.,pathways that mediate host-microbiota interactions, e.g., host immunesystem pathways or bacteriocyte pathways, e.g., a gene encoding aprotein listed in Table 7, Table 8, or Table 9, all with reference toaccession number provided).

iv. Target Genes and Proteins

Any of the modulating agents described herein can be used to alter(e.g., increase or decrease) gene expression, alter (e.g., increase ordecrease) a target protein activity, and/or alter function in the hostor a microorganism resident in the host. Proteins or genes that areinvolved in a variety of processes may be targeted, including any of thefunctional classes listed in Table 6.

TABLE 6 Functional classes of target genes Functional class EffectHomeo- Downregulation or knockout of these genes will disrupt innerstasis target microorganism homeostatic balance generating in the host acellular malfunction and therefore a decrease in their fitness. Inform-Downregulation or knockout of these genes will stop or slow ationprotein synthesis therefore affecting proliferation of themicroorganism. This in turn will generate in the host a cellularmalfunction and therefore a decrease in their fitness. Meta-Downregulation or knockout of these genes will stop the bolismcatabolism and anabolism of nutrients affecting proliferation of themicroorganism. This in turn will generate in the host a cellularmalfunction and therefore a decrease in their fitness. TransportDownregulation or knockout of these genes will stop or decrease thetransport of amino acids or their precursors therefore affectingmicroorganism survival. This in turn will generate in the host acellular malfunction and therefore a decrease in their fitness.

(a) Target Microbial Genes and Proteins

Any of the modulating agents described herein can be used to alter geneexpression or target proteins in a microorganism resident in the host.In some instances, the modulating agent (e.g., an antibody) directlytargets a protein in a microorganism resident in the host, including anyone of the proteins listed in Table 7. In other instances, themodulating agent (e.g., nucleic acid, e.g., RNAi) alters gene expression(e.g., increases or decreases gene expression) in a microorganismresident in the host, including genes that encode any of the proteinslisted in Table 7, in comparison to a host organism to which themodulating agent has not been administered.

TABLE 7 Target bacterial proteins Functional Sequence class Protein nameAccession No. Organism Homeostasis chaperonin GroEL NP_239860.1 Buchneraaphidicola str. APS (Acyrthosiphon pisum) Homeostasis DnaK proteinNP_239985.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Homeostasis heat shock protein GrpE1 NP_240076.1 Buchnera aphidicolastr. APS (Acyrthosiphon pisum) Homeostasis ATP-dependent proteaseATP-binding NP_240382.1 Buchnera subunit aphidicola str. APS(Acyrthosiphon pisum) Homeostasis heat shock protein GrpE2 NP_240015.1Buchnera aphidicola str. APS (Acyrthosiphon pisum) Informationmethionine aminopeptidase NP_240059.1 Buchnera aphidicola str. APS(Acyrthosiphon pisum) Information 30S ribosomal protein S1 NP_240132.1Buchnera aphidicola str. APS (Acyrthosiphon pisum) Information DnaJprotein NP_239984.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information polynucleotide NP_240191.1 Buchneraphosphorylase/polyadenylase aphidicola str. APS (Acyrthosiphon pisum)Information DNA gyrase subunit B NP_239852.1 Buchnera aphidicola str.APS (Acyrthosiphon pisum) Information tryptophanyl-tRNA synthetaseNP_240343.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information threonyl-tRNA synthetase NP_239957.1 Buchnera aphidicolastr. APS (Acyrthosiphon pisum) Information alanyl-tRNA synthetaseNP_240220.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information asparaginyl-tRNA synthetase NP_240178.1 Buchnera aphidicolastr. APS (Acyrthosiphon pisum) Information tRNA(guanine-N1)-methyltransferase NP_240213.1 Buchnera aphidicola str. APS(Acyrthosiphon pisum) Information 50S ribosomal protein L30 NP_240313.1Buchnera aphidicola str. APS (Acyrthosiphon pisum) Informationreplicative DNA helicase NP_240352.2 Buchnera aphidicola str. APS(Acyrthosiphon pisum) Information glycyl-tRNA synthetase subunit alphaNP_239968.2 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information A/G-specific adenine glycosylase NP_240358.1 Buchneraaphidicola str. APS (Acyrthosiphon pisum) Information tRNA pseudouridine55 synthase NP_240193.1 Buchnera aphidicola str. APS (Acyrthosiphonpisum) Information methionyl-tRNA synthetase NP_239942.1 Buchneraaphidicola str. APS (Acyrthosiphon pisum) Information lysyl-tRNAsynthetase NP_240385.1 Buchnera aphidicola str. APS (Acyrthosiphonpisum) Information glutaminyl-tRNA synthetase NP_240227.1 Buchneraaphidicola str. APS (Acyrthosiphon pisum) Information DNA polymerase INP_240243.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information ribosomal large subunit pseudouridine NP_240167.1 Buchnerasynthase C aphidicola str. APS (Acyrthosiphon pisum) Informationhistidyl-tRNA synthetase NP_240112.1 Buchnera aphidicola str. APS(Acyrthosiphon pisum) Information DNA polymerase III subunits gammaNP_240292.1 Buchnera and tau aphidicola str. APS (Acyrthosiphon pisum)Information tRNA modification GTPase TrmE NP_239858.2 Buchneraaphidicola str. APS (Acyrthosiphon pisum) Information DNA polymerase IIIbeta chain NP_239853.1 Buchnera aphidicola str. APS (Acyrthosiphonpisum) Information RNA polymerase sigma factor RpoD NP_239892.1 Buchneraaphidicola str. APS (Acyrthosiphon pisum) Information arginyl-tRNAsynthetase NP_240071.1 Buchnera aphidicola str. APS (Acyrthosiphonpisum) Information seryl-tRNA synthetase NP_240135.1 Buchnera aphidicolastr. APS (Acyrthosiphon pisum) Information DNA polymerase III alphachain NP_240067.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information aspartyl-tRNA synthetase NP_240138.1 Buchnera aphidicolastr. APS (Acyrthosiphon pisum) Information leucyl-tRNA synthetaseNP_240256.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Information phenylalanyl-tRNA synthetase beta NP_239962.1 Buchnera chainaphidicola str. APS (Acyrthosiphon pisum) Metabolism5-methyltetrahydropteroyltriglutamate-- NP_239871.1 Buchnerahomocysteine methyltransferase aphidicola str. APS (Acyrthosiphon pisum)Metabolism bifunctional aspartokinase NP_240025.1 Buchnera I/homeserinedehydrogenase I aphidicola str. APS (Acyrthosiphon pisum) Metabolismsulfate adenylate transferase subunit 1 NP_240235.1 Buchnera aphidicolastr. APS (Acyrthosiphon pisum) Metabolism adenylosuccinate synthetaseNP_240370.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum)Metabolism phosphoadenosine phosphosulfate NP_240238.1 Buchnerareductase aphidicola str. APS (Acyrthosiphon pisum) Metabolismphosphoserine aminotransferase NP_240134.1 Buchnera aphidicola str. APS(Acyrthosiphon pisum) Metabolism F0F1 ATP synthase subunit gammaNP_239849.1 Buchnera aphidicola str. APS (Acyrthosiphon pisum) Transportcell division inhibitor MinC NP_240149.1 Buchnera aphidicola str. APS(Acyrthosiphon pisum) Transport 23S rRNA m(2)G2445 NP_240181.1 Buchneramethyltransferase aphidicola str. APS (Acyrthosiphon pisum) TransporttRNA uridine 5- NP_239843.1 Buchnera carboxymethylaminomethyl aphidicolastr. modification enzyme GidA APS (Acyrthosiphon pisum)

(b) Target Genes and Proteins in Hosts

Any of the modulating agents described herein can be used to alter geneexpression or target proteins in the host. In some instances, themodulating agent (e.g., an antibody) directly targets a protein in thehost, including any one of the proteins listed in Table 8 or Table 9. Inother instances, the modulating agent (e.g., nucleic acid, e.g., RNAi)targets a gene in the host, including genes that encode any of theproteins listed in Table 8 or Table 9. For example, the nucleic acidsdescribed herein can be used to alter (e.g., increase or decrease) geneexpression in a host (e.g., genes that regulate bacteriocyte function ordevelopment (e.g., bacteriocyte regulatory peptides) or genes thatregulate the immune system) including, but not limited to, any of thegenes listed in Table 8 and Table 9, in comparison to a host organism towhich the modulating agent has not been administered.

TABLE 8 Proteins that regulate bacteriocyte function Sequence Functionalaccession class Protein name number Organism Homeostasis ns1 bindingprotein ACYPI000701- Acyrthosiphon PA pisum Homeostasis Ubxtranscription factor ACYPI001856- Acyrthosiphon RA pisum MetabolismATPase ACYPI002584- Acyrthosiphon PA pisum Metabolism Succinicsemialdehyde ACYPI002063- Acyrthosiphon dehydrogenase isoform 1 PA pisumMetabolism Vacuolar proton atpases isoform 1 ACYPI007445- AcyrthosiphonPA pisum Metabolism Membrane alanyl aminopeptidase N ACYPI006675-Acyrthosiphon PA pisum Metabolism Protease m1 zinc metalloproteaseACYPI009427- Acyrthosiphon PA pisum Metabolism Zinc metalloproteaseACYPI000580- Acyrthosiphon PA pisum Metabolism Purine biosynthesisprotein 6, pur6 ACYPI010114- Acyrthosiphon PA pisum Metabolism Myoinositol monophosphatase ACYPI009018- Acyrthosiphon PA pisum MetabolismPhenylalanine hydroxylase ACYPI007803- Acyrthosiphon PA pisum Metabolism4-nitrophenylphosphatase isoform 1 ACYPI005939- Acyrthosiphon PA pisumMetabolism Cytosolic purine 5-nucleotidase ACYPI007730- Acyrthosiphon PApisum Metabolism Amine oxidase ACYPI006507- Acyrthosiphon PA pisumMetabolism Phosphoserine aminotransferase ACYPI004666- Acyrthosiphon PApisum Metabolism 4-Hydroxybutyrate CoA-transferase ACYPI001782-Acyrthosiphon PA pisum Metabolism Dihydropyrimidine dehydrogenaseACYPI004747- Acyrthosiphon PA pisum Metabolism Glycine dehydrogenase,ACYPI005060- Acyrthosiphon mitochondrial PA pisum MetabolismRibose-phosphate ACYPI006288- Acyrthosiphon pyrophosphokinase 1,putative PA pisum Metabolism Phosphoserine phosphatase isoform 1ACYPI000304- Acyrthosiphon PA pisum Metabolism Adeninephosphoribosyltransferase ACYPI003436- Acyrthosiphon PA pisum MetabolismPantothenate kinase 4 (Pantothenic ACYPI003518- Acyrthosiphon acidkinase 4) (hPanK4) PA pisum Metabolism Phosphoenolpyruvate carboxykinaseACYPI001978- Acyrthosiphon PA pisum Metabolism Cystathionine beta-lyase,partial ACYPI000593- Acyrthosiphon PA pisum Metabolism Putative5-nucleotidase, partial ACYPI002452- Acyrthosiphon PA pisum MetabolismZipper CG15792-PD ACYPI004129- Acyrthosiphon PA pisum MetabolismAconitase ACYPI008211- Acyrthosiphon PA pisum MetabolismProphenoloxidase ACYPI001367- Acyrthosiphon PA pisum MetabolismGlycinamide ribonucleotide ACYPI009448- Acyrthosiphonsynthetase-aminoimidazole PA pisum ribonucleotide synthetase-glycinamide ribonucleotide transformylase, partial Metabolism Aldehydedehydrogenase ACYPI003925- Acyrthosiphon PA pisum MetabolismMetalloprotease ACYPI008675- Acyrthosiphon PA pisum MetabolismProphenoloxidase ACYPI004484- Acyrthosiphon PA pisum Metabolism5-aminoimidazole-4-carboxamide ACYPI008919- Acyrthosiphon ribonucleotideformyltransferase/IMP PA pisum cyclohydrolase Metabolism Sec24B protein,putative ACYPI005848- Acyrthosiphon PA pisum Metabolism Gmp synthaseACYPI006177- Acyrthosiphon PA pisum Metabolism mCG117402 ACYPI000180-Acyrthosiphon PA pisum Metabolism Lambda-crystallin ACYPI001738-Acyrthosiphon PA pisum Metabolism Glyoxylate/hydroxypyruvateACYPI001693- Acyrthosiphon reductase PA pisum MetabolismFructose-1,6-bisphosphatase ACYPI002694- Acyrthosiphon PA pisumMetabolism Imaginal disk growth factor ACYPI001365- Acyrthosiphon PApisum Metabolism Lysosomal alpha-mannosidase ACYPI000371- Acyrthosiphon(mannosidase alpha class 2b PA pisum member 1) Metabolism 5-oxoprolinase(ATP-hydrolysing) ACYPI004211- Acyrthosiphon PA pisum MetabolismAspartate ammonia lyase ACYPI006003- Acyrthosiphon PA pisum MetabolismAldo-keto reductase ACYPI005685- Acyrthosiphon PA pisum MetabolismAminomethyltransferase ACYPI002795- Acyrthosiphon PA pisum RegulatoryBacteriocyte-specific cysteine rich ACYPI32128 Acyrthosiphon proteinsBCR family, peptide BCR1 pisum Regulatory Bacteriocyte-specific cysteinerich ACYPI38738 Acyrthosiphon proteins BCR family, peptide BCR2 pisumRegulatory Bacteriocyte-specific cysteine rich ACYPI44142 Acyrthosiphonproteins BCR family, peptide BCR3 pisum Regulatory Bacteriocyte-specificcysteine rich ACYPI49532 Acyrthosiphon proteins BCR family, peptide BCR6pisum Regulatory Bacteriocyte-specific cysteine rich ACYPI45157Acyrthosiphon proteins BCR family, peptide BCR8 pisum RegulatorySecreted proteins SP family, peptide ACYPI008389 Acyrthosiphon SP1 pisumRegulatory Secreted proteins SP family, peptide ACYPI000294Acyrthosiphon SP2 pisum Regulatory Secreted proteins SP family, peptideACYPI005168 Acyrthosiphon SP3 pisum Regulatory Secreted proteins SPfamily, peptide ACYPI009984 Acyrthosiphon SP4 pisum Regulatory Secretedproteins SP family, peptide ACYPI004796 Acyrthosiphon SP5a pisumRegulatory Secreted proteins SP family, peptide ACYPI001839Acyrthosiphon SP6 pisum Signaling Glean peptide GLEAN_28598 ACYPI48598-Acyrthosiphon PA pisum Signaling Glean peptide GLEAN_33885 ACYPI53885-Acyrthosiphon PA pisum Signaling past-1 ACYPI007266- Acyrthosiphon PApisum Transport Putative vacuolar ATP synthase ACYPI006090-Acyrthosiphon subunit E isoform 1 PA pisum Transport Vacuolar ATPsynthase subunit H ACYPI002312- Acyrthosiphon PA pisum TransportVacuolar ATPase subunit C ACYPI006545- Acyrthosiphon PA pisum TransportVacuolar ATP synthase 16 kDa ACYPI003545- Acyrthosiphon proteolipidsubunit (Ductin) PA pisum (VHA16K) Transport Potassium/chloridesymporter, ACYPI000507- Acyrthosiphon putative, partial PA pisumTransport Cationic amino acid transporter ACYPI008904- Acyrthosiphon PApisum

In some instances, the methods or compositions provided herein may beeffective to decrease the host's resistance to parasites or pathogens(e.g., fungal, bacterial, or viral pathogens or parasites) in comparisonto a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to decrease the host's resistance to a pathogenor parasite (e.g., fungal, bacterial, or viral pathogens; or parasiticmites) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or greater than 100% relative to a reference level (e.g., a levelfound in a host that does not receive a modulating agent).

In some instances, the modulating agent is effective to alter the innateimmune system of a host to indirectly change microbial diversity in thehost relative to a host organism to which the modulating agent has notbeen administered. Invertebrates exhibit multiple immune reactions, someof which are homologous to immune mechanisms found in mammals. Generalprinciples of innate immunity in insects have been summarized by otherreviews (Lemaitre et al., Annu. Rev. Immunol. 25:697-743, 2007; Charrouxet al., Fly 4:40-47, 2010; Ganesan et al., Curr. Top. Microbiol.Immunol. 349:25-60, 2011; Chambers et al., Curr. Opin. Immunol.24:10-14, 2012). For example, in D. melanogaster, there are two majorinducible responses enabling local immunity at the intestinal epithelialcell layer: production of AMPs and synthesis of reactive oxygen species(ROS). While both of these induced responses might be seen as classicalresistance mechanisms, they both include negative feedback loops andmodulatory components, which can confer host tolerance toward thecommensal gut microbiota.

Colonization of the gut by commensal bacteria could induce immunepriming events resulting in the activation or alteration of the immuneresponse not only toward recurrent colonization of commensal bacteria,but also against pathogens. Gut microbiota is essential not only forpriming the immune system of the host to bacteria, e.g., mosquitoes toPlasmodium, but also for eliciting the priming response uponrechallenging the hosts with the bacteria. The bacteria-dependentpriming response in mosquitoes is characterized by differentiation ofprohemocytes into granulocytes and the presence of increased numbers ofcirculating granulocytes with changed morphology and binding properties.In another example, tsetse fly bacterial symbionts, including thegut-inhabiting Gammaproteobacterium S. glossinidius, are essentialduring larval development in order that the adult flies could present atrypanosome-refractory phenotype. In this case, the bacteria seem toinfluence the formation and integrity of the adult peritrophic matrix,thereby indirectly regulating the fly's ability to detect and respond tothe presence of trypanosomes.

In some instances, an immune response is modulated by production of amodulating agent. For example, in the systemic immune response of D.melanogaster, Toll and IMD are the two major signaling pathways inducingantimicrobial peptide (AMP) production. Upon pathogen exposure, only theIMD pathway is active and triggers a local AMP response. Activationoccurs by binding different variants of bacterial peptidoglycan (PGN) toextra- or intracellular epithelial receptors belonging to thepeptidoglycan recognition protein (PGRP) family. The protein Pirksequesters specific PGN-binding receptors (PGRP-LC) in the cytoplasm,thereby reducing the number of these receptors localizing to the cellsurface and retarding IMD pathway signaling. PGRP-LE ensures immunetolerance to the commensal microbiota via the up-regulation of amidasesand Pirk. Downstream signaling via the IMD pathway results in activationof the transcription factor Relish, which in turn induces expression ofseveral AMPs and other immunity-related genes. PGRP-LE induces aRelish-dependent immune response to pathogenic bacteria. PGRP-LE alsoensures immune tolerance to the commensal microbiota via theup-regulation of amidases and Pirk.

The homeobox transcription factor Caudal specifically represses AMP genetranscription in the gut by binding to promoter regions.Caudal-deficiency causes a constitutive AMP production to occur and ashift in the gut microbiota. In one embodiment, an IMD pathway isinactivated to restrict expression of one or more AMPs and/or otherimmunity-related genes, e.g., caudal deficiency, thereby activating animmune response to a gut microbiota.

In some instances, exposure to pathogens in the gut triggers thegeneration of ROS via the membrane-associated dual oxidase (DUOX)system. For example, in D. melanogaster, PGN-dependent andPGN-independent signaling pathways produce ROS that causes oxidativestress not only on the bacteria but also on the hosts epithelial cells.D. melanogaster eliminates excessive ROS by activating immune responsivecatalases. This catalase production results in increased tolerance, dueto a decrease in self-harm caused by the bacteria-induced immuneresponse, possibly through locally restricted catalase activity, e.g.,to the proximity of the epithelial surface. In one embodiment, immuneresponsive catalases are inactivated or repressed to maintain ROSproduction via the DUOX system and oxidative stress via PGN-dependentand PGN-independent signaling pathways to activate an immune response toa gut microbiota.

In some instances, the modulating agent is effective to alter (e.g.,increase or decrease) gene expression in a host to increase or decreasethe host's immune system response or immunoregulatory signaling, e.g.,immune system response to microorganisms resident in the host (e.g.,microorganisms resident in host bacteriocytes) in comparison to a hostorganism to which the modulating agent has not been administered.Nonlimiting examples of immune system related genes/proteins inbacteriocytes are shown in Table 9.

TABLE 9 Immunoregulatory proteins that modulate bacteriocytes SequenceFunctional accession class Protein name number Organism Regulatory BicD(Protein bicaudal D) CG6605 D. melanogaster Regulatory PGRP SA(Peptidoglycan CG11709 D. melanogaster recognition protein) RegulatoryRelish (transcription factor) CG11992 D. melanogaster Regulatory Pirk(poor Imd response upon CG15678 D. melanogaster knock-in) RegulatoryDUOX (Dual oxidase) CG3131 D. melanogaster Regulatory p38c (p38c MAPkinase) CG33338 D. melanogaster Regulatory MKP3 (Dual phosphatase)CG14080 D. melanogaster Regulatory CanB (calcineurin B) CG4209 D.melanogaster Regulatory Plad (phospholipase D) CG12110 D. melanogasterRegulatory Cact (cactus) CG5848 D. melanogaster Regulatory DIF (Dorsalrelated immunity CG6794 D. melanogaster factor) Regulatory dIAP2(Drosophila Inhibitor of CG8293 D. melanogaster APoptosis2) RegulatoryToll (Toll Interacting Protein) CG5490 D. melanogaster Regulatory gnbp1(Gram Negative Binding CG6895 D. melanogaster Protein1) Regulatory LysC(c-type lysozyme) CR9111 D. melanogaster Regulatory imd (immunedeficiency) CG5576 D. melanogaster Regulatory Diap2 (Death-associatedinhibitor CG8293 D. melanogaster of apoptosis 2) Regulatory ecsitCG10610 D. melanogaster

v. Bacteria as Modulating Agents

In some instances, the modulating agent described herein includes one ormore bacteria. Numerous bacteria are useful in the compositions andmethods described herein. In some instances, the agent is a bacterialspecies endogenously found in the host. In some instances, the bacterialmodulating agent is an endosymbiotic bacterial species. In someinstances, the bacterial modulating agent is a pathogen in the host.Non-limiting examples of bacteria that may be used as modulating agentsinclude all bacterial species described herein in Section II of thedetailed description and those listed in Table 1. For example, themodulating agent may be a bacterial species from any bacterial phylapresent in host (e.g., insect, mollusk, or nematode) guts, includingGammaproteobacteria, Alphaproteobacteria, Betaproteobacteria,Bacteroidetes, Firmicutes (e.g., Lactobacillus and Bacillus spp.),Clostridia, Actinomycetes, Spirochetes, Verrucomicrobia, andActinobacteria.

In some instances, the bacteria may be used as a modulating agent tostimulate an immune response in the host that leads to a decrease in thelevel, diversity, or metabolism of one or more microorganisms residentin the host. In some instances, the bacteria are delivered as livebacterial cells (e.g., E. coli cells). Alternatively, the bacteria maybe delivered as heat-killed bacterial cells (e.g., heat-killed E. colicells). In other instances, the bacteria may be delivered as lysates(e.g., prepared from lysed whole cells), or fractions thereof.

In some instances, the modulating agent includes genetically modified ortransformed bacteria as described herein. In some instances, thebacteria are genetically modified. For example, the bacteria may bemodified via the introduction of genetic material into a bacterium usingstandard methods in the art (e.g., transduction, transformation, orconjugation), thereby modifying or altering the cellular physiology,and/or biochemistry of the bacterium. Through the introduction ofgenetic material, the modified bacteria may acquire new functions orproperties. In some instances, the genetically modified bacteria mayproduce and secrete a modulating agent described herein. For example,genetically modified bacteria may produce polypeptides, small molecules,or nucleic acids that target specific host endosymbionts or othermicroorganisms resident in the host. In some instances, the geneticallymodified bacteria may be used to produce a product that stimulates ahost immune response.

In some instances, the genetically engineered or transformed bacteriaare provided to impart new functionalities to the host. Newfunctionalities may include, for example, the ability to degradepesticides (e.g., insecticides, molluscicides, or nematicides; e.g., apesticide listed in Table 11), plant allelochemicals, or producenutrients. For example, genetically modified bacteria may be generatedfrom naturally occurring bacteria isolated from pesticide (e.g.,insecticide, molluscicide, or nematicides; e.g., a pesticide listed inTable 11) resistant pests, e.g. Burkholderia strains from the insect R.pedestris. When bacteria from insecticide resistant pests are culturedwith commensal bacteria isolated from a host of interest, e.g., bees,genes imparting insecticide resistance (e.g., the ability to use theinsecticide as a carbon source) may be transferred to the commensalbacteria of the host of interest, e.g., bees. The genetically modifiedbacteria are then reintroduced into the host, e.g., bees, andinsecticide resistant insects are selected by breeding them in anenvironment rich in the insecticide. Any bacterial phyla that arecommonly present in a host may be modified to have a new functionality,including, but not limited to, Gammaproteobacteria, Alphaproteobacteria,Betaproteobacteria, Bacteroidetes, Firmicutes including Lactobacillusand Bacillus species, Clostridia, Actinomycetes, Spirochetes,Verrucomicrobia, Actinobacteria, and others.

The bacterium may be genetically modified to decrease the fitness of thehost or to kill the host. In some instances, a bacterium may begenetically modified to improve its survival in host cells, to increaseits toxicity to the host cell, and/or to provide a function thatdecreases the fitness of the host (e.g., decreased resistance to apesticide, e.g., a pesticide listed in Table 11). In some instances, thebacterium is modified to no longer synthesize an essential molecule thatit typically provides to the host cell. In some instances, the bacteriumis genetically modified so that the population of the bacteria isincreased to a level that negatively impacts the host, e.g., byexcessive utilization of essential materials or machinery or bycompeting with beneficial microorganisms resident in the host. In someinstances, the bacteria only kill the host following further humanintervention.

The genetically modified bacteria described herein can be generatedusing any methods known in the art. For example, methods for thedelivery of nucleic acids to bacteria include, but are not limited to,different chemical, electrochemical and biological approaches, forexample, heat shock transformation, electroporation, transfection, forexample liposome-mediated transfection, DEAE-Dextran-mediatedtransfection or calcium phosphate transfection. In some instances, anucleic acid construct, for example, an expression construct includingat least one nucleic acid sequence is introduced into the bacteria usinga vehicle, or vector, for transferring genetic material. Vectors fortransferring genetic material to bacteria are well known to those ofskill in the art and include, for example, plasmids, artificialchromosomes, and viral vectors. Methods for the construction of nucleicacid constructs, including expression constructs including constitutiveor inducible heterologous promoters, knockout and knockdown constructs,as well as methods and vectors for the delivery of a nucleic acid ornucleic acid construct to bacteria are well known to those of skill inthe art, and are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001; Amberg et al., Methods in YeastGenetics: A Cold Spring Harbor Laboratory Course Manual, Cold SpringHarbor Laboratory Press, 2005; Abelson et al., Guide to Yeast Geneticsand Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series,194), Academic Press, 2004; Guthrie et al., Guide to Yeast Genetics andMolecular and Cell Biology, 1st edition, Part B, Volume 350 (Methods inEnzymology, Vol 350), Academic Press, 2002; Stephanopoulos et al.,Metabolic Engineering: Principles and Methodologies, 1st edition,Academic Press, 1998; and Smolke, The Metabolic Pathway EngineeringHandbook: Fundamentals, 1st edition, CRC Press, 2009, all of which areincorporated by reference herein in their entireties.

vi. Fungi as Modulating Agents

In some instances, the modulating agent described herein includes one ormore fungi. Numerous fungi are useful in the compositions and methods.For example, the fungal modulating agent may be yeast such asSaccharomyces cerevisiae or Pichia pastoris.

In some instances, the fungi (e.g., S. cerevisiae or P. pastoris) may beused as modulating agents to stimulate an immune response in the hostthat leads to a decrease in the levels, diversity, or metabolism of oneor more microorganisms (e.g., bacteria or fungi) resident in the host.In some instances, the fungi are delivered as live fungal cells (e.g.,P. pastoris cells, see Example 15). Alternatively, the fungi may bedelivered as heat-killed fungal cells (e.g., heat-killed S. cerevisiaecells). In other instances, the fungi may be delivered as lysates (e.g.,prepared from lysed whole cells), or fractions thereof.

In some instances, the modulating agent includes a genetically modifiedor transformed fungus as described herein. In some instances, the fungusis genetically modified. For example, the fungus may be modified via theintroduction of genetic material into a fungus using standard methods inthe art, thereby modifying or altering the cellular physiology and/orbiochemistry of the fungus. Through the introduction of geneticmaterial, the modified fungus may acquire new functions or properties.In some instances, the genetically modified fungus may produce andsecrete a modulating agent described herein. In some instances, thegenetically modified fungus may be used to produce a product thatstimulates a host immune response.

In some instances, the genetically engineered or transformed fungus isprovided to impart new functionalities to the host. New functionalitiesmay include, for example, the ability to degrade pesticides (e.g.,insecticides), plant allelochemicals, or produce nutrients. Any fungalphyla that are commonly present in a host may be genetically modified tohave a new functionality, including, but not limited to, Candida,Metschnikowia, Debaromyces, Scheffersomyces shehatae and Scheffersomycesstipites, Starmerella, Pichia, Trichosporon, Cryptococcus, Pseudozyma,and yeast-like symbionts from the subphylum Pezizomycotina (e.g.,Symbiotaphrina bucneri and Symbiotaphrina kochii).

As illustrated by Example 15, yeast, such as P. pastoris, can be used asa modulating agent that stimulates a host immune response (e.g., in aninsect, e.g., an aphid) that, in turn, alters the activity, levels, ormetabolism of endosymbiotic bacteria, such as a Buchnera spp., residentin the host and thereby modulates (e.g., decreases) the fitness of thehost.

vii. Modifications to Modulating Agents

In some instances, the nucleic acid molecule, peptide, polypeptide, orantibody molecule can be modified. For example, the modification can bea chemical modification, e.g., conjugation to a marker, e.g.,fluorescent marker or a radioactive marker. In other examples, themodification can include conjugation or operational linkage to a moietythat enhances the stability, delivery, targeting, bioavailability, orhalf-life of the agent, e.g., a lipid, a glycan, a polymer (e.g., PEG),a cation moiety.

(a) Fusions

Any of the modulating agents described herein may be fused or linked toan additional moiety. In some instances, the modulating agent includes afusion of one or more additional moieties (e.g., 1 additional moiety, 2,3, 4, 5, 6, 7, 8, 9, 10, or more additional moieties). In someinstances, the additional moiety is any one of the modulating agentsdescribed herein (e.g., a peptide, polypeptide, small molecule, orantibiotic). Alternatively, the additional moiety may not act asmodulating agent itself but may instead serve a secondary function. Forexample, the additional moiety may to help the modulating agent access,bind, or become activated at a target site in the host (e.g., at a hostgut or a host bacteriocyte) or at a target microorganism resident in thehost.

In some instances, the additional moiety may help the modulating agentpenetrate a target host cell or target microorganism resident in thehost. For example, the additional moiety may include a cell penetratingpeptide. Cell penetrating peptides (CPPs) may be natural sequencesderived from proteins; chimeric peptides that are formed by the fusionof two natural sequences; or synthetic CPPs, which are syntheticallydesigned sequences based on structure-activity studies. In someinstances, CPPs have the capacity to ubiquitously cross cellularmembranes (e.g., prokaryotic and eukaryotic cellular membranes) withlimited toxicity. Further, CPPs may have the capacity to cross cellularmembranes via energy-dependent and/or independent mechanisms, withoutthe necessity of a chiral recognition by specific receptors.Non-limiting examples of CPPs are listed in Table 10.

TABLE 10 Examples of Cell Penetrating Peptides (CPPs) Peptide OriginSequence Protein-derived Penetratin Antennapedia RQIKIWFQNRRMKWKK (SEQID NO: 82) Tat peptide Tat GRKKRRQRRRPPQ (SEQ ID NO: 83) pVEC CadherinLLIILRRRIRKQAHAHSK (SEQ ID NO: 84) Chimeric TransportanGalanine/Mastoparan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 85) MPGHIV-gp41/SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 86)Pep-1 HIV-reverse KETWWETWWTEWSQPKKKRKV transcriptase/SV40 T- (SEQ IDNO: 87) antigen Synthetic Polyarginines Based on Tat peptide (R)_(n); 6< n < 12 MAP de novo KLALKLALKALKAALKLA (SEQ ID NO: 88) R₆W₃ Based onpenetratin RRWWRRWRR (SEQ ID NO: 89)

In some instances, the additional moiety is a peptide nucleic acid(PNAs). Peptide nucleic acids (PNAs) include one or more nucleic acidside chains linked to an amide backbone. One or more amino acid units inthe PNA have an amide containing backbone, e.g., aminoethyl-glycine,similar to a peptide backbone, with a nucleic acid side chain in placeof the amino acid side chain. PNAs are known to hybridize complementaryDNA and RNA with higher affinity than their oligonucleotidecounterparts. This character of PNA not only makes the polypeptide ofthe invention a stable hybrid with the nucleic acid side chains, but atthe same time, the neutral backbone and hydrophobic side chains resultin a hydrophobic unit within the polypeptide. Examples of PNA moietiesinclude a molecule that includes a peptide, such as a CPP (e.g., anamino acid sequence having at least at least 80% (e.g., 80%, 90%, 95%,97%, 99%, or greater) identity to SEQ ID NO: 106) and a nucleotidesequence having at least 80% (e.g., 80%, 90%, 95%, 97%, 99%, or greater)identity to the sequence of any one of SEQ ID NOs: 105 or 151-153.

The nucleic acid side chain includes, but is not limited to, a purine ora pyrimidine side chain such as adenine, cytosine, guanine, thymine, anduracil. In one instances, the nucleic acid side chain includes anucleoside analog, such as 5-fluorouracil; 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,4-methylbenzimidazole, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil (acp³ U),2,6-diaminopurine, 3-nitropyrrole, inosine, thiouridine, queuosine,wyosine, diaminopurine, isoguanine, isocytosine, diaminopyrimidine,2,4-difluorotoluene, isoquinoline, pyrrolo[2,3-β]pyridine, and anyothers that can base pair with a purine or a pyrimidine side chain.

In other instances, the additional moiety helps the modulating agentbind a target microorganism (e.g., a fungi or bacterium) resident in thehost. The additional moiety may include one or more targeting domains.In some instances, the targeting domain may target the modulating agentto one or more microorganisms (e.g., bacterium or fungus) resident inthe gut of the host. In some instances, the targeting domain may targetthe modulating agent to a specific region of the host (e.g., host gut orbacteriocyte) to access microorganisms that are generally present insaid region of the host. For example, the targeting domain may targetthe modulating agent to the foregut, midgut, or hindgut of the host. Inother instances, the targeting domain may target the modulating agent toa bacteriocyte in the host and/or one or more specific bacteria residentin a host bacteriocyte.

(b) Pre- or Pro-Domains

In some instances, the modulating agent may include a pre- or pro-aminoacid sequence. For example, the modulating agent may be an inactiveprotein or peptide that can be activated by cleavage orpost-translational modification of a pre- or pro-sequence. In someinstances, the modulating agent is engineered with an inactivating pre-or pro-sequence. For example, the pre- or pro-sequence may obscure anactivation site on the modulating agent, e.g., a receptor binding site,or may induce a conformational change in the modulating agent. Thus,upon cleavage of the pre- or pro-sequence, the modulating agent isactivated.

Alternatively, the modulating agent may include a pre- or pro-smallmolecule, e.g., an antibiotic. The modulating agent may be an inactivesmall molecule described herein that can be activated in a targetenvironment inside the host. For example, the small molecule may beactivated upon reaching a certain pH in the host gut.

(c) Linkers

In instances where the modulating agent is connected to an additionalmoiety, the modulating agent may further include a linker. For example,the linker may be a chemical bond, e.g., one or more covalent bonds ornon-covalent bonds. In some instances, the linker may be a peptidelinker (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, ormore amino acids longer). The linker maybe include any flexible, rigid,or cleavable linkers described herein.

A flexible peptide linker may include any of those commonly used in theart, including linkers having sequences having primarily Gly and Serresidues (“GS” linker). Flexible linkers may be useful for joiningdomains that require a certain degree of movement or interaction and mayinclude small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) aminoacids.

Alternatively, a peptide linker may be a rigid linker. Rigid linkers areuseful to keep a fixed distance between moieties and to maintain theirindependent functions. Rigid linkers may also be useful when a spatialseparation of the domains is critical to preserve the stability orbioactivity of one or more components in the fusion. Rigid linkers may,for example, have an alpha helix-structure or Pro-rich sequence,(XP)_(n), with X designating any amino acid, preferably Ala, Lys, orGlu.

In yet other instances, a peptide linker may be a cleavable linker. Insome instances, linkers may be cleaved under specific conditions, suchas the presence of reducing reagents or proteases. In vivo cleavablelinkers may utilize the reversible nature of a disulfide bond. Oneexample includes a thrombin-sensitive sequence (e.g., PRS) between twoCys residues. In vitro thrombin treatment of CPRSC results in thecleavage of the thrombin-sensitive sequence, while the reversibledisulfide linkage remains intact. Such linkers are known and described,e.g., in Chen et al., Adv. Drug Deliv. Rev. 65(10):1357-1369, 2013.Cleavage of linkers in fusions may also be carried out by proteases thatare expressed in vivo under conditions in specific cells or tissues ofthe host or microorganisms resident in the host. In some instances,cleavage of the linker may release a free functional, modulating agentupon reaching a target site or cell.

Fusions described herein may alternatively be linked by a linkingmolecule, including a hydrophobic linker, such as a negatively chargedsulfonate group; lipids, such as a poly (—CH2—) hydrocarbon chains, suchas polyethylene glycol (PEG) group, unsaturated variants thereof,hydroxylated variants thereof, amidated or otherwise N-containingvariants thereof, non-carbon linkers; carbohydrate linkers;phosphodiester linkers, or other molecule capable of covalently linkingtwo or more molecules, e.g., two modulating agents. Non-covalent linkersmay be used, such as hydrophobic lipid globules to which the modulatingagent is linked, for example, through a hydrophobic region of themodulating agent or a hydrophobic extension of the modulating agent,such as a series of residues rich in leucine, isoleucine, valine, orperhaps also alanine, phenylalanine, or even tyrosine, methionine,glycine, or other hydrophobic residue. The modulating agent may belinked using charge-based chemistry, such that a positively chargedmoiety of the modulating agent is linked to a negative charge of anothermodulating agent or an additional moiety.

IV. Formulations and Compositions

The compositions described herein may be formulated either in pure form(e.g., the composition contains only the modulating agent) or togetherwith one or more additional agents (such as excipient, delivery vehicle,carrier, diluent, stabilizer, etc.) to facilitate application ordelivery of the compositions. Examples of suitable excipients anddiluents include, but are not limited to, lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, saline solution, syrup,methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesiumstearate, and mineral oil.

In some instances, the composition includes a delivery vehicle orcarrier. In some instances, the delivery vehicle includes an excipient.Exemplary excipients include, but are not limited to, solid or liquidcarrier materials, solvents, stabilizers, slow-release excipients,colorings, and surface-active substances (surfactants). In someinstances, the delivery vehicle is a stabilizing vehicle. In someinstances, the stabilizing vehicle includes a stabilizing excipient.Exemplary stabilizing excipients include, but are not limited to,epoxidized vegetable oils, antifoaming agents, e.g. silicone oil,preservatives, viscosity regulators, binding agents and tackifiers. Insome instances, the stabilizing vehicle is a buffer suitable for themodulating agent. In some instances, the composition ismicroencapsulated in a polymer bead delivery vehicle. In some instances,the stabilizing vehicle protects the modulating agent against UV and/oracidic conditions. In some instances, the delivery vehicle contains a pHbuffer. In some instances, the composition is formulated to have a pH inthe range of about 4.5 to about 9.0, including for example pH ranges ofabout any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5to about 7.0.

Depending on the intended objectives and prevailing circumstances, thecomposition may be formulated into emulsifiable concentrates, suspensionconcentrates, directly sprayable or dilutable solutions, coatablepastes, diluted emulsions, spray powders, soluble powders, dispersiblepowders, wettable powders, dusts, granules, encapsulations in polymericsubstances, microcapsules, foams, aerosols, carbon dioxide gaspreparations, tablets, resin preparations, paper preparations, nonwovenfabric preparations, or knitted or woven fabric preparations. In someinstances, the composition is a liquid. In some instances, thecomposition is a solid. In some instances, the composition is anaerosol, such as in a pressurized aerosol can. In some instances, thecomposition is present in the waste (such as feces) of the pest. In someinstances, the composition is present in or on a live pest.

In some instances, the delivery vehicle is the food or water of thehost. In other instances, the delivery vehicle is a food source for thehost. In some instances, the delivery vehicle is a food bait for thehost. In some instances, the composition is a comestible agent consumedby the host. In some instances, the composition is delivered by the hostto a second host, and consumed by the second host. In some instances,the composition is consumed by the host or a second host, and thecomposition is released to the surrounding of the host or the secondhost via the waste (such as feces) of the host or the second host. Insome instances, the modulating agent is included in food bait intendedto be consumed by a host or carried back to its colony.

In some instances, the delivery vehicle is a bacterial vector. Themodulating agent can be incorporated in a bacterial vector using anysuitable cloning methods and reagents known in the art, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.“Bacterial vector” as used herein refers to any genetic element, such asplasmids, bacteriophage vectors, transposons, cosmids, and chromosomes,which is capable of replication inside bacterial cells and which iscapable of transferring genes between cells. Exemplary bacterial vectorsinclude, but are not limited to, lambda vector system gtl 1, gt WES.tB,Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177,pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKCIOI, SV 40,pBluescript II SK+/− or KS+/−(see “Stratagene Cloning Systems” Catalog,Stratagene, La Jolla, Calif., 1993), pQE, pIH821, pGEX, pET series (seeStudier et al., “Use of T7 RNA Polymerase to Direct Expression of ClonedGenes,” Gene Expression Technology, Vol. 185, 1990), and any derivativesthereof.

Each bacterial vector may encode one or more modulating agents. In someinstances, the bacterial vector includes a nucleic acid moleculeencoding a polypeptide to be expressed in the target symbiotic bacteriumor a host bacterium. In some instances, the bacterial vector includes anucleic acid molecule encoding a bacteriocin to be expressed in thetarget bacterium. In some instances, the bacterial vector furtherincludes one or more regulatory elements, such as promoters, terminationsignals, and transcription and translation elements. In some instances,the regulatory sequence is operably linked to a nucleic acid encoding agene (e.g., any of the nucleic acids described herein) to be expressedin the target symbiotic bacterium.

In some instances, the bacterial vector is introduced into a bacteriumto be consumed by the host or a member in the colony of the host. Insome instances, the bacterium is the target symbiotic bacterium. In someinstances, the bacterium is a naturally occurring bacterium of the gutof the host, or a genetically modified derivative thereof, which can beeasily introduced to the host through ingestion. Exemplary bacteria foruse in carrying the bacterial vector include, but are not limited to,Proteobacter, including the genus Pseudomonas; Actinobacter, includingPriopionibacterium and Corynebacterium; Firmicutes, including the anyspecies of the genera Mycoplasma, Bacillus, Streptococcus,Staphylococcus; Fibrobacteres; Spirochaetes, including Treponema andBorrelia; Bacteroides, including the genera Bacteroides andFlavobacterium. Also suitable are any bacteria of theEnterobacteriaceae, including the genus Serratia, including, but notlimited to S. marcescens, S. entomophila, S. proteamaculans; any speciesof Enterobacter, including, but not limited to, E. cloacae, E.aerogenes, E. dissolvens, E. agglomerans, E. hafiiiae; and any speciesbelonging to the following genera: Citrobacter, Escherichia, Klebsiella,Kluyvera, Panotea, Proteus, Salmonella, Xenorhabdus, and Yokenella.

In some instances, the modulating agent may make up about 0.1% to about100% of the composition, such as any one of about 0.01% to about 100%,about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%,about 10% to about 50%, about 50% to about 99%, or about 0.1% to about90% of active ingredients (e.g., a polypeptide, nucleic acid, smallmolecule, or combinations thereof). In some instances, the compositionincludes at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more active ingredients (e.g., apolypeptide, nucleic acid, small molecule, or combinations thereof). Insome instances, the concentrated agents are preferred as commercialproducts, the final user normally uses diluted agents, which have asubstantially lower concentration of active ingredient.

Any of the formulations described herein may be used in the form of abait, a coil, an electric mat, a smoking preparation, a fumigant, or asheet.

i. Liquid Formulations

The compositions provided herein may be in a liquid formulation. Liquidformulations are generally mixed with water, but in some instances maybe used with crop oil, diesel fuel, kerosene or other light oil as acarrier. The amount of active ingredient often ranges from about 0.5 toabout 80 percent by weight.

An emulsifiable concentrate formulation may contain a liquid activeingredient, one or more petroleum-based solvents, and an agent thatallows the formulation to be mixed with water to form an emulsion. Suchconcentrates may be used in agricultural, ornamental and turf, forestry,structural, food processing, livestock, and public health pestformulations. These may be adaptable to application equipment from smallportable sprayers to hydraulic sprayers, low-volume ground sprayers,mist blowers, and low-volume aircraft sprayers. Some active ingredientsare readily dissolve in a liquid carrier. When mixed with a carrier,they form a solution that does not settle out or separate, e.g., ahomogenous solution. Formulations of these types may include an activeingredient, a carrier, and one or more other ingredients. Solutions maybe used in any type of sprayer, indoors and outdoors.

In some instances, the composition may be formulated as an invertemulsion. An invert emulsion is a water-soluble active ingredientdispersed in an oil carrier. Invert emulsions require an emulsifier thatallows the active ingredient to be mixed with a large volume ofpetroleum-based carrier, usually fuel oil. Invert emulsions aid inreducing drift. With other formulations, some spray drift results whenwater droplets begin to evaporate before reaching target surfaces; as aresult the droplets become very small and lightweight. Because oilevaporates more slowly than water, invert emulsion droplets shrink lessand more active ingredient reaches the target. Oil further helps toreduce runoff and improve rain resistance. It further serves as asticker-spreader by improving surface coverage and absorption. Becausedroplets are relatively large and heavy, it is difficult to get thoroughcoverage on the undersides of foliage. Invert emulsions are mostcommonly used along rights-of-way where drift to susceptible non-targetareas can be a problem.

A flowable or liquid formulation combines many of the characteristics ofemulsifiable concentrates and wettable powders. Manufacturers use theseformulations when the active ingredient is a solid that does notdissolve in either water or oil. The active ingredient, impregnated on asubstance such as clay, is ground to a very fine powder. The powder isthen suspended in a small amount of liquid. The resulting liquid productis quite thick. Flowables and liquids share many of the features ofemulsifiable concentrates, and they have similar disadvantages. Theyrequire moderate agitation to keep them in suspension and leave visibleresidues, similar to those of wettable powders.

Flowables/liquids are easy to handle and apply. Because they areliquids, they are subject to spilling and splashing. They contain solidparticles, so they contribute to abrasive wear of nozzles and pumps.Flowable and liquid suspensions settle out in their containers. Becauseflowable and liquid formulations tend to settle, packaging in containersof five gallons or less makes remixing easier.

Aerosol formulations contain one or more active ingredients and asolvent. Most aerosols contain a low percentage of active ingredients.There are two types of aerosol formulations—the ready-to-use typecommonly available in pressurized sealed containers and those productsused in electrical or gasoline-powered aerosol generators that releasethe formulation as a smoke or fog.

Ready to use aerosol formulations are usually small, self-containedunits that release the formulation when the nozzle valve is triggered.The formulation is driven through a fine opening by an inert gas underpressure, creating fine droplets. These products are used ingreenhouses, in small areas inside buildings, or in localized outdoorareas. Commercial models, which hold five to 5 pounds of activeingredient, are usually refillable.

Smoke or fog aerosol formulations are not under pressure. They are usedin machines that break the liquid formulation into a fine mist or fog(aerosol) using a rapidly whirling disk or heated surface.

ii. Dry or Solid Formulations

Dry formulations can be divided into two types: ready-to-use andconcentrates that must be mixed with water to be applied as a spray.Most dust formulations are ready to use and contain a low percentage ofactive ingredients (less than about 10 percent by weight), plus a veryfine, dry inert carrier made from talc, chalk, clay, nut hulls, orvolcanic ash. The size of individual dust particles varies. A few dustformulations are concentrates and contain a high percentage of activeingredients. Mix these with dry inert carriers before applying. Dustsare always used dry and can easily drift to non-target sites.

iii. Granule or Pellet Formulations

In some instances, the composition is formulated as granules. Granularformulations are similar to dust formulations, except granular particlesare larger and heavier. The coarse particles may be made from materialssuch as clay, corncobs, or walnut shells. The active ingredient eithercoats the outside of the granules or is absorbed into them. The amountof active ingredient may be relatively low, usually ranging from about0.5 to about 15 percent by weight. Granular formulations are most oftenused to apply to the soil, insects, mollusks, or nematodes living in thesoil, or absorption into plants through the roots. Granular formulationsare sometimes applied by airplane or helicopter to minimize drift or topenetrate dense vegetation. Once applied, granules may release theactive ingredient slowly. Some granules require soil moisture to releasethe active ingredient. Granular formulations also are used to controllarval mosquitoes and other aquatic pests. Granules are used inagricultural, structural, ornamental, turf, aquatic, right-of-way, andpublic health (biting insect) pest-control operations.

In some instances, the composition is formulated as pellets. Most pelletformulations are very similar to granular formulations; the terms areused interchangeably. In a pellet formulation, however, all theparticles are the same weight and shape. The uniformity of the particlesallows use with precision application equipment.

iv. Powders

In some instances, the composition is formulated as a powder. In someinstances, the composition is formulated as a wettable powder. Wettablepowders are dry, finely ground formulations that look like dusts. Theyusually must be mixed with water for application as a spray. A fewproducts, however, may be applied either as a dust or as a wettablepowder—the choice is left to the applicator. Wettable powders have about1 to about 95 percent active ingredient by weight; in some cases morethan about 50 percent. The particles do not dissolve in water. Theysettle out quickly unless constantly agitated to keep them suspended.They can be used for most pest problems and in most types of sprayequipment where agitation is possible. Wettable powders have excellentresidual activity. Because of their physical properties, most of theformulation remains on the surface of treated porous materials such asconcrete, plaster, and untreated wood. In such cases, only the waterpenetrates the material.

In some instances, the composition is formulated as a soluble powder.Soluble powder formulations look like wettable powders. However, whenmixed with water, soluble powders dissolve readily and form a truesolution. After they are mixed thoroughly, no additional agitation isnecessary. The amount of active ingredient in soluble powders rangesfrom about 15 to about 95 percent by weight; in some cases more thanabout 50 percent. Soluble powders have all the advantages of wettablepowders and none of the disadvantages, except the inhalation hazardduring mixing.

In some instances, the composition is formulated as a water-dispersiblegranule. Water-dispersible granules, also known as dry flowables, arelike wettable powders, except instead of being dust-like, they areformulated as small, easily measured granules. Water-dispersiblegranules must be mixed with water to be applied. Once in water, thegranules break apart into fineparticles similar to wettable powders. Theformulation requires constant agitation to keep it suspended in water.The percentage of active ingredient is high, often as much as 90 percentby weight. Water-dispersible granules share many of the same advantagesand disadvantages of wettable powders, except they are more easilymeasured and mixed. Because of low dust, they cause less inhalationhazard to the applicator during handling

v. Bait

In some instances, the composition includes a bait. The bait can be inany suitable form, such as a solid, paste, pellet or powdered form. Thebait can also be carried away by the host back to a population of saidhost (e.g., a colony or hive). The bait can then act as a food sourcefor other members of the colony, thus providing an effective modulatingagent for a large number of hosts and potentially an entire host colony.

The baits can be provided in a suitable “housing” or “trap.” Suchhousings and traps are commercially available and existing traps can beadapted to include the compositions described herein. The housing ortrap can be box-shaped for example, and can be provided in pre-formedcondition or can be formed of foldable cardboard for example. Suitablematerials for a housing or trap include plastics and cardboard,particularly corrugated cardboard. The inside surfaces of the traps canbe lined with a sticky substance in order to restrict movement of thehost once inside the trap. The housing or trap can contain a suitabletrough inside which can hold the bait in place. A trap is distinguishedfrom a housing because the host cannot readily leave a trap followingentry, whereas a housing acts as a “feeding station” which provides thehost with a preferred environment in which they can feed and feel safefrom predators.

vi. Attractants

In some instances, the composition includes an attractant (e.g., achemoattractant). The attractant may attract an adult host or immaturehost (e.g., larva) to the vicinity of the composition. Attractantsinclude pheromones, a chemical that is secreted by an animal, especiallya host (e.g., insect, mollusk, or nematode), which influences thebehavior or development of others of the same species. Other attractantsinclude sugar and protein hydrolysate syrups, yeasts, and rotting meat.Attractants also can be combined with an active ingredient and sprayedonto foliage or other items in the treatment area.

Various attractants are known which influence host behavior as a host'ssearch for food, oviposition or mating sites, or mates. Attractantsuseful in the methods and compositions described herein include, forexample, eugenol, phenethyl propionate, ethyldimethylisobutyl-cyclopropane carboxylate, propylbenszodioxancarboxylate, cis-7,8-epoxy-2-methyloctadecane,trans-8,trans-O-dodecadienol, cis-9-tetradecenal (withcis-11-hexadecenal), trans-11-tetradecenal, cis-11-hexadecenal,(Z)-11,12-hexadecadienal, cis-7-dodecenyl acetate, cis-8-dodecenyulacetate, cis-9-dodecenyl acetate, cis-9-tetradecenyl acetate,cis-11-tetradecenyl acetate, trans-11-tetradecenyl acetate (withcis-11), cis-9,trans-11-tetradecadienyl acetate (with cis-9,trans-12),cis-9,trans-1 2-tetradecadienyl acetate, cis-7,cis-11-hexadecadienylacetate (with cis-7,trans-11), cis-3,cis-13-octadecadienyl acetate,trans-3,cis-13-octadecadienyl acetate, anethole and isoamyl salicylate.

Means other than chemoattractants may also be used to attract a host(e.g., insect, mollusk, or nematode), including lights in variouswavelengths or colors.

vii. Nanocapsules/Microencapsulation/Liposomes

In some instances, the composition is provided in a microencapsulatedformulation. Microencapsulated formulations are mixed with water andsprayed in the same manner as other sprayable formulations. Afterspraying, the plastic coating breaks down and slowly releases the activeingredient.

viii. Carriers

Any of the compositions described herein may be formulated to includethe modulating agent described herein and an inert carrier. Such carriercan be a solid carrier, a liquid carrier, a gel carrier, and/or agaseous carrier. In certain instances, the carrier can be a seedcoating. The seed coating is any non-naturally occurring formulationthat adheres, in whole or part, to the surface of the seed. Theformulation may further include an adjuvant or surfactant. Theformulation can also include one or more modulating agents to enlargethe action spectrum.

A solid carrier used for formulation includes finely-divided powder orgranules of clay (e.g. kaolin clay, diatomaceous earth, bentonite,Fubasami clay, acid clay, etc.), synthetic hydrated silicon oxide, talc,ceramics, other inorganic minerals (e.g., sericite, quartz, sulfur,activated carbon, calcium carbonate, hydrated silica, etc.), a substancewhich can be sublimated and is in the solid form at room temperature(e.g., 2,4,6-triisopropyl-1,3,5-trioxane, naphthalene,p-dichlorobenzene, camphor, adamantan, etc.); wool; silk; cotton; hemp;pulp; synthetic resins (e.g., polyethylene resins such as low-densitypolyethylene, straight low-density polyethylene and high-densitypolyethylene; ethylene-vinyl ester copolymers such as ethylene-vinylacetate copolymers; ethylene-methacrylic acid ester copolymers such asethylene-methyl methacrylate copolymers and ethylene-ethyl methacrylatecopolymers; ethylene-acrylic acid ester copolymers such asethylene-methyl acrylate copolymers and ethylene-ethyl acrylatecopolymers; ethylene-vinylcarboxylic acid copolymers such asethylene-acrylic acid copolymers; ethylene-tetracyclododecenecopolymers; polypropylene resins such as propylene homopolymers andpropylene-ethylene copolymers; poly-4-methylpentene-1, polybutene-1,polybutadiene, polystyrene; acrylonitrile-styrene resins; styreneelastomers such as acrylonitrile-butadiene-styrene resins,styrene-conjugated diene block copolymers, and styrene-conjugated dieneblock copolymer hydrides; fluororesins; acrylic resins such aspoly(methyl methacrylate); polyamide resins such as nylon 6 and nylon66; polyester resins such as polyethylene terephthalate, polyethylenenaphthalate, polybutylene terephthalate, andpolycyclohexylenedimethylene terephthalate; polycarbonates, polyacetals,polyacrylsulfones, polyarylates, hydroxybenzoic acid polyesters,polyetherimides, polyester carbonates, polyphenylene ether resins,polyvinyl chloride, polyvinylidene chloride, polyurethane, and porousresins such as foamed polyurethane, foamed polypropylene, or foamedethylene, etc.), glasses, metals, ceramics, fibers, cloths, knittedfabrics, sheets, papers, yarn, foam, porous substances, andmultifilaments.

A liquid carrier may include, for example, aromatic or aliphatichydrocarbons (e.g., xylene, toluene, alkylnaphthalene,phenylxylylethane, kerosine, gas oil, hexane, cyclohexane, etc.),halogenated hydrocarbons (e.g., chlorobenzene, dichloromethane,dichloroethane, trichloroethane, etc.), alcohols (e.g., methanol,ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethyleneglycol, etc.), ethers (e.g., diethyl ether, ethylene glycol dimethylether, diethylene glycol monomethyl ether, diethylene glycol monoethylether, propylene glycol monomethyl ether, tetrahydrofuran, dioxane,etc.), esters (e.g., ethyl acetate, butyl acetate, etc.), ketones (e.g.,acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone,etc.), nitriles (e.g., acetonitrile, isobutyronitrile, etc.), sulfoxides(e.g., dimethyl sulfoxide, etc.), amides (e.g., N,N-dimethylformamide,N,N-dimethylacetamide, cyclic imides (e.g. N-methylpyrrolidone)alkylidene carbonates (e.g., propylene carbonate, etc.), vegetable oil(e.g., soybean oil, cottonseed oil, etc.), vegetable essential oils(e.g., orange oil, hyssop oil, lemon oil, etc.), or water.

A gaseous carrier may include, for example, butane gas, flon gas,liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide gas.

ix. Adjuvants

In some instances, the composition provided herein may include anadjuvant. Adjuvants are chemicals that do not possess activity.Adjuvants are either pre-mixed in the formulation or added to the spraytank to improve mixing or application or to enhance performance. Theyare used extensively in products designed for foliar applications.Adjuvants can be used to customize the formulation to specific needs andcompensate for local conditions. Adjuvants may be designed to performspecific functions, including wetting, spreading, sticking, reducingevaporation, reducing volatilization, buffering, emulsifying,dispersing, reducing spray drift, and reducing foaming. No singleadjuvant can perform all these functions, but compatible adjuvants oftencan be combined to perform multiple functions simultaneously.

Among nonlimiting examples of adjuvants included in the formulation arebinders, dispersants and stabilizers, specifically, for example, casein,gelatin, polysaccharides (e.g., starch, gum arabic, cellulosederivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars,synthetic water-soluble polymers (e.g., polyvinyl alcohol,polyvinylpyrrolidone, polyacrylic acid, etc.), PAP (acidic isopropylphosphate), BHT (2,6-di-t-butyl-4-methylphenol), BHA (a mixture of2-t-butyl-4-methoxyphenol and 3-t-butyl-4-methoxyphenol), vegetableoils, mineral oils, fatty acids and fatty acid esters.

x. Surfactants

In some instances, the composition provided herein includes asurfactant. Surfactants, also called wetting agents and spreaders,physically alter the surface tension of a spray droplet. For aformulation to perform its function properly, a spray droplet must beable to wet the foliage and spread out evenly over a leaf. Surfactantsenlarge the area of formulation coverage, thereby increasing the pest'sexposure to the chemical. Surfactants are particularly important whenapplying a formulation to waxy or hairy leaves. Without proper wettingand spreading, spray droplets often run off or fail to cover leafsurfaces adequately. Too much surfactant, however, can cause excessiverunoff and reduce efficacy.

Surfactants are classified by the way they ionize or split apart intoelectrically charged atoms or molecules called ions. A surfactant with anegative charge is anionic. One with a positive charge is cationic, andone with no electrical charge is nonionic. Formulation activity in thepresence of a nonionic surfactant can be quite different from activityin the presence of a cationic or anionic surfactant. Selecting the wrongsurfactant can reduce the efficacy of a pesticide product and injure thetarget plant. Anionic surfactants are most effective when used withcontact pesticides (pesticides that control the pest by direct contactrather than being absorbed systemically). Cationic surfactants shouldnever be used as stand-alone surfactants because they usually arephytotoxic.

Nonionic surfactants, often used with systemic pesticides, helppesticide sprays penetrate plant cuticles. Nonionic surfactants arecompatible with most pesticides, and most EPA-registered pesticides thatrequire a surfactant recommend a nonionic type. Adjuvants include, butare not limited to, stickers, extenders, plant penetrants, compatibilityagents, buffers or pH modifiers, drift control additives, defoamingagents, and thickeners.

Among nonlimiting examples of surfactants included in the compositionsdescribed herein are alkyl sulfate ester salts, alkyl sulfonates, alkylaryl sulfonates, alkyl aryl ethers and polyoxyethylenated productsthereof, polyethylene glycol ethers, polyvalent alcohol esters and sugaralcohol derivatives.

xi. Combinations

In formulations and in the use forms prepared from these formulations,the modulating agent may be in a mixture with other active compounds,such as pesticidal agents (e.g., insecticides, sterilants, acaricides,nematicides, molluscicides, or fungicides; e.g., a pesticide listed inTable 11), attractants, growth-regulating substances, or herbicides. Asused herein, the term “pesticidal agent” refers to any substance ormixture of substances intended for preventing, destroying, repelling, ormitigating any pest. A pesticide can be a chemical substance orbiological agent used against pests including insects, mollusks,pathogens, weeds, nematodes, and microbes that compete with humans forfood, destroy property, spread disease, or are a nuisance. The term“pesticidal agent” may further encompass other bioactive molecules suchas antibiotics, antivirals pesticides, antifungals, antihelminthics,nutrients, pollen, sucrose, and/or agents that stun or slow insectmovement.

In instances where the modulating agent is applied to plants, a mixturewith other known compounds, such as herbicides, fertilizers, growthregulators, safeners, semiochemicals, or else with agents for improvingplant properties is also possible.

V. Delivery

A host described herein can be exposed to any of the compositionsdescribed herein in any suitable manner that permits delivering oradministering the composition to the host invertebrate (e.g., insect,mollusk, or nematode). The modulating agent may be delivered eitheralone or in combination with other active or inactive substances and maybe applied by, for example, spraying, microinjection, through plants,pouring, dipping, in the form of concentrated liquids, gels, solutions,suspensions, sprays, powders, pellets, briquettes, bricks and the like,formulated to deliver an effective concentration of the modulatingagent. Amounts and locations for application of the compositionsdescribed herein are generally determined by the habits of the host, thelifecycle stage at which the microorganisms of the host can be targetedby the modulating agent, the site where the application is to be made,and the physical and functional characteristics of the modulating agent.The modulating agents described herein may be administered to the hostinvertebrate (e.g., insect, mollusk, or nematode) by oral ingestion, butmay also be administered by means which permit penetration through thecuticle or penetration of the host (e.g., insect, mollusk, or nematode)respiratory system.

In some instances, the invertebrate host (e.g., insect, mollusk, ornematode) can be simply “soaked” or “sprayed” with a solution includingthe modulating agent. Alternatively, the modulating agent can be linkedto a food component (e.g., comestible) of the invertebrate host (e.g.,insect, mollusk, or nematode) for ease of delivery and/or in order toincrease uptake of the modulating agent by the host. Methods for oralintroduction include, for example, directly mixing a modulating agentwith the host's food, spraying the modulating agent in the host'shabitat or field, as well as engineered approaches in which a speciesthat is used as food is engineered to express a modulating agent, thenfed to the host to be affected. In some instances, for example, themodulating agent composition can be incorporated into, or overlaid onthe top of, the host's diet. For example, the modulating agentcomposition can be sprayed onto a field of crops which a host inhabits.

In some instances, the composition is sprayed directly onto a plante.g., crops, by e.g., backpack spraying, aerial spraying, cropspraying/dusting etc. In instances where the modulating agent isdelivered to a plant, the plant receiving the modulating agent may be atany stage of plant growth. For example, formulated modulating agents canbe applied as a seed-coating or root treatment in early stages of plantgrowth or as a total plant treatment at later stages of the crop cycle.In some instances, the modulating agent may be applied as a topicalagent to a plant, such that the host invertebrate (e.g., insect,mollusk, or nematode) ingests or otherwise comes in contact with theplant upon interacting with the plant.

Further, the modulating agent may be applied (e.g., in the soil in whicha plant grows, or in the water that is used to water the plant) as asystemic agent that is absorbed and distributed through the tissues of aplant or animal host, such that a host invertebrate (e.g., insect,mollusk, or nematode) feeding thereon will obtain an effective dose ofthe modulating agent. In some instances, plants or food organisms may begenetically transformed to express the modulating agent such that a hostfeeding upon the plant or food organism will ingest the modulatingagent.

Delayed or continuous release can also be accomplished by coating themodulating agent or a composition with the modulating agent(s) with adissolvable or bioerodable coating layer, such as gelatin, which coatingdissolves or erodes in the environment of use, to then make themodulating agent available, or by dispersing the agent in a dissolvableor erodable matrix. Such continuous release and/or dispensing meansdevices may be advantageously employed to consistently maintain aneffective concentration of one or more of the modulating agentsdescribed herein in a specific host habitat.

Alternatively, the modulating agent is expressed in a bacterial orfungal cell and the bacterial or fungal cell is taken up or eaten by thehost invertebrate (e.g., insect, mollusk, or nematode) species. Bacteriaor fungi can be engineered to produce any of the modulating agentsdescribed herein. In other instances, a virus such as a baculoviruswhich specifically infects host invertebrates (e.g., insect, mollusk, ornematode) may also be used. This ensures safety for mammals, especiallyhumans and animals, since the virus will not infect mammals.

The modulating agent can also be incorporated into the medium in whichthe host invertebrate (e.g., insect, mollusk, or nematode) grows, lives,reproduces, feeds, or infests. For example, a modulating agent can beincorporated into a food container, feeding station, protectivewrapping, or a hive. For some applications the modulating agent may bebound to a solid support for application in powder form or in a “trap”or “feeding station.” As an example, for applications where thecomposition is to be used in a trap or as bait for a particular hostinvertebrate (e.g., insect, mollusk, or nematode), the compositions mayalso be bound to a solid support or encapsulated in a time-releasematerial. For example, the compositions described herein can beadministered by delivering the composition to at least one habitat wherean agricultural pest (e.g., aphid) grows, lives, reproduces, or feeds.

i. Engineered Plants

The terms “genetically engineered plant” or “transgenic plant” refer toa plant cell or a plant that expresses a modulating agent. Thetransgenic plants are also meant to include progeny (decedent,offspring, etc.) of any generation of such a transgenic plant or a seedof any generation of all such transgenic plants wherein said progeny orseed includes a modulating agent.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed. For example, the constructs can be introduced in a varietyof forms including, but not limited to as a strand of DNA, in a plasmid,or in an artificial chromosome. The introduction of the constructs intothe target plant cells can be accomplished by a variety of techniques,including, but not limited to Agrobacterium-mediated transformation,electroporation, microinjection, microprojectile bombardmentcalcium-phosphate-DNA co-precipitation or liposome-mediatedtransformation of a heterologous nucleic acid. The transformation of theplant is preferably permanent, i.e. by integration of the introducedexpression constructs into the host plant genome, so that the introducedconstructs are passed onto successive plant generations.

Any plant species may be transformed to create a transgenic plant. Thetransgenic plant may be a dicotyledonous plant or a mono-cotyledonousplant. For example and without limitation, transgenic plants of thecompositions and methods described herein may be derived from any of thefollowing diclotyledonous plant families: Leguminosae, including plantssuch as pea, alfalfa and soybean; Umbelliferae, including plants such ascarrot and celery; Solanaceae, including the plants such as tomato,potato, aubergine, tobacco, and pepper; Cruciferae, particularly thegenus Brassica, which includes plant such as oilseed rape, beet,cabbage, cauliflower and broccoli); and Arabidopsis thaliana;Compositae, which includes plants such as lettuce; Malvaceae, whichincludes cotton; Fabaceae, which includes plants such as peanut, and thelike. Transgenic plants of the invention may be derived frommonocotyle-donous plants, such as, for example, wheat, barley, sorghum,millet, rye, triticale, maize, rice, oats, switchgrass, miscanthus, andsugarcane. Transgenic plants of the invention are also embodied as treessuch as apple, pear, quince, plum, cherry, peach, nectarine, apricot,papaya, mango, and other woody species including coniferous anddeciduous trees such as poplar, pine, sequoia, cedar, oak, willow, andthe like.

Any promoter capable of driving expression in the plant of interest maybe used. The promoter may be native or analogous or foreign orheterologous to the plant host. The choice of promoters to be includeddepends upon several factors, including, but not limited to, efficiency,selectability, inducibility, desired expression level, and cell- ortissue-preferential expression. It is a routine matter for one of skillin the art to modulate the expression of a sequence by appropriatelyselecting and positioning promoters and other regulatory regionsrelative to that sequence.

Promoters active in photosynthetic tissue in order to drivetranscription in green tissues such as leaves and stems are ofparticular interest. Most suitable are promoters that drive expressiononly or predominantly in such tissues. The promoter may conferexpression constitutively throughout the plant, or differentially withrespect to the green tissues, or differentially with respect to thedevelopmental stage of the green tissue in which expression occurs, orin response to external stimuli.

Examples of such promoters include the ribulose-1,5-bisphosphatecarboxylase (RbcS) promoters such as the RbcS promoter from easternlarch (Larix laricina), the pine cab6 promoter (Yamamoto et al., PlantCell Physiol. 35:773-778, 1994), the Cab-1 gene promoter from wheat(Fejes et al., Plant Mol. Biol. 15:921-932, 1990), the CAB-1 promoterfrom spinach (Lubberstedt et al., Plant Physiol. 104:997-1006, 1994),the cab1 R promoter from rice (Luan et al., Plant Cell 4:971-981, 1992),the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuokaet al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993), the tobaccoLhcb1*2 promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), theArabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al.Planta 196:564-570, 1995), and thylakoid membrane protein promoters fromspinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Otherpromoters that drive transcription in stems, leafs and green tissue aredescribed in U.S. Patent Publication No. 2007/0006346. The TrpA promoteris a pith preferred promoter and has been described in U.S. Pat. No.6,018,104.

A maize gene encoding phosphoenol carboxylase (PEPC) has been describedby Hudspeth et al. (Plant Molec. Biol. 12:579-589, 1989). Using standardmolecular biological techniques the promoter for this gene can be usedto drive the expression of any gene in a green tissue-specific manner intransgenic plants.

In some other instances, inducible promoters may be desired. Induciblepromoters drive transcription in response to external stimuli such aschemical agents or environmental stimuli. For example, induciblepromoters can confer transcription in response to hormones such asgiberellic acid or ethylene, or in response to light or drought.

VI. Screening

Any of the modulating agents described herein may be isolated from ascreening assay, wherein a library of modulating agents (e.g., a mixtureof variants of a starting modulating agent) is screened for modulatingagents (e.g., modulating agent variants) that are effective to alter themicrobiota of a host (e.g., insect/mollusk/nematode) and therebymodulate (e.g., increase or decrease) host fitness.

For example, the screening assays provided herein may be effective toidentify one or more modulating agents (e.g., a polypeptide, nucleicacid, small molecule, or combinations thereof) that target symbioticmicroorganisms resident in the host and thereby decrease the fitness ofthe host. For example, the identified modulating agent (e.g., apolypeptide, nucleic acid, small molecule, or combinations thereof) maybe effective to decrease the viability of pesticide- orallelochemical-degrading microorganisms (e.g., bacteria e.g., abacterium that degrades a pesticide listed in Table 11), therebyincreasing the host's sensitivity to a pesticide (e.g., sensitivity to apesticide listed in Table 11) or allelochemical agent.

Alternatively, a screening assay may be used to identify a modulatingagent effective to increase host fitness (e.g., insect, mollusk, ornematode fitness). For example, the screening assay may be used toidentify one or more modulating agents that target specificmicroorganisms and/or specific hosts. Further, the screening assays maybe used to identify modulating agents that provide one or moremicroorganisms with enhanced functionalities. For example, the screeningassay may be effective to isolate modulating agents that provide one ormore microorganisms with an enhanced ability to metabolize (e.g.,degrade) a pesticide (e.g., insecticide, e.g., neonicotinoid) or plantallelochemical (e.g., caffeine, soyacystatin, fenitrothion,monoterpenes, diterpene acids, or phenolic compounds (e.g., tannins,flavonoids)). Delivery and colonization of an isolated microorganism inthe host may increase the host's resistance to the pesticide or plantallelochemical, thereby increasing host fitness. The methods may also beuseful for the isolation of modulating agents that providemicroorganisms with an enhanced ability to colonize any of the hostsdescribed herein.

TABLE 11 Pesticides Aclonifen Acetamiprid Alanycarb AmidosulfuronAminocyclopyrachlor Amisulbrom Anthraquinone Asulam, sodium saltBenfuracarb Bensulide beta-HCH; beta-BCH Bioresmethrin Blasticidin-SBorax; disodium tetraborate Boric acid Bromoxynil heptanoate Bromoxyniloctanoate Carbosulfan Chlorantraniliprole Chlordimeform ChlorfluazuronChlorphropham Climbazole Clopyralid Copper (II) hydroxide CyflufenamidCyhalothrin Cyhalothrin, gamma Decahydrate Diafenthiuron DimefuronDimoxystrobin Dinotefuran Diquat dichloride Dithianon E-PhosphamidonEPTC Ethaboxam Ethirimol Fenchlorazole-ethyl Fenothiocarb FentirothionFenpropidin Fluazolate Flufenoxuron Flumetralin FluxapyroxadFuberidazole Glufosinate-ammonium Glyphosate Group: Borax, borate salts(see Group: Paraffin oils, Mineral Halfenprox Imiprothrin ImidaclopridIpconazole Isopyrazam Isopyrazam Lenacil Magnesium phosphideMetaflumizone Metazachlor Metazachlor Metobromuron MetoxuronMetsulfuron-methyl Milbemectin Naled Napropamide Nicosulfuron NitenpyramNitrobenzene o-phenylphenol oils Oxadiargyl Oxycarboxin Paraffin oilPenconazole Pendimethalin Penflufen Penflufen PentachlorbenzenePenthiopyrad Penthiopyrad Pirimiphos-methyl Prallethrin ProfenofosProquinazid Prothiofos Pyraclofos Pyrazachlor Pyrazophos PyridabenPyridalyl Pyridiphenthion Pyrifenox Quinmerac Rotenone Sedaxane SedaxaneSilafluofen Sintofen Spinetoram Sulfoxaflor Temephos thioclopridThiamethoxam Tolfenpyrad Tralomethrin Tributyltin compounds TridiphaneTriflumizole Validamycin Zinc phosphide

EXAMPLES

The following is an example of the methods of the invention. It isunderstood that various other embodiments may be practiced, given thegeneral description provided above.

Example 1: Generation of a cDNA Library from Corn Leaf Aphid Larvae(Rhopalosiphum maidis)

This Example demonstrates the production of a cDNA library from cornleaf aphid larvae (Rhopalosiphum maidis).

Experimental Design:

To generate the library, RNA from 0.9 g whole first-instar larvae (4 to5 days post-hatch; held at 16° C.) is purified using the followingphenol/TRI REAGENT®-based method (MOLECULAR RESEARCH CENTER, Cincinnati,Ohio). Larvae are homogenized at room temperature in a 15 mL homogenizerwith 10 mL of TRI REAGENT® until a homogenous suspension is obtained.Following 5 min incubation at room temperature, the homogenate isdispensed into 1.5 mL microfuge tubes (1 mL per tube), 200 μL ofchloroform is added, and the mixture is vigorously shaken for 15seconds. After allowing the extraction to sit at room temperature for 10min, the phases are separated by centrifugation at 12,000×g at 4° C. Theupper phase (including about 0.6 mL) is carefully transferred intoanother sterile 1.5 mL tube, and an equal volume of room temperatureisopropanol is added. After incubation at room temperature for 5 to 10min, the mixture is centrifuged 8 min at 12,000×g (4° C. or 25° C.).

The supernatant is carefully removed and discarded, and the RNA pelletis washed twice by vortexing with 75% ethanol, with recovery bycentrifugation for 5 min at 7,500×g (4° C. or 25° C.) after each wash.The ethanol is carefully removed, the pellet is allowed to air-dry for 3to 5 min, and then is dissolved in nuclease-free sterile water. RNAconcentration is determined by measuring the absorbance (A) at 260 nmand 280 nm. The RNA extracted is stored at −80° C. until furtherprocessed, and RNA quality is determined by running an aliquot through a1% agarose gel.

The larval total RNA is converted into a cDNA library using randompriming. The larval cDNA library is sequenced at ½ plate scale by GS FLX454 Titanium™ series chemistry at EUROFINS MWG Operon, which results inover 600,000 reads with an average read length of 348 bp. 350,000 readsare assembled into over 50,000 contigs. Both the unassembled reads andthe contigs are converted into BLASTable databases using the publiclyavailable program, FORMATDB (available from NCBI).

Example 2: Production and Purification of Bcr1 dsRNA

This Example demonstrates the production and purification of a syntheticdsRNA from a cDNA library.

Experimental Design:

Bcr1 gene (ACYP132128) is an essential gene for bacteriocyte regulationand function in insects. Bcr1 cDNA is prepared from the larval total RNAdescribed in Example 1 and for Bcr1 dsRNA synthesis prepared by PCRusing the primer pairs: Forward 5′-aaactgctgcatggctttct-3′ (SEQ ID NO:90) and reverse 5′-acaggcctttcaggctttta-3′ (SEQ ID NO: 91). For thetarget gene region, two separate PCR amplifications are performed. Thefirst PCR amplification introduces a T7 promoter sequence at the 5′ end((TTAATACGACTCACTATAGGGAGA; SEQ ID NO: 92) of the amplified sensestrands. The second reaction incorporates the T7 promoter sequence atthe 5′ ends of the antisense strands. The two PCR amplified fragmentsfor each region of the target Bcr1 gene are then mixed in equal amounts,and the mixture is used as a transcription template for dsRNAproduction. Double-stranded RNA for insect bioassay is synthesized andpurified using an AMBION® MEGASCRIPT® RNAi kit following themanufacturer's instructions (INVITROGEN) or HiScribe® T7 In VitroTranscription Kit following the manufacturer's instructions (New EnglandBiolabs, Ipswich, Mass.). The concentration of Bcr1 dsRNA is measuredusing a NANODROP™ 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington,Del.) and the purified Bcr1 dsRNA is prepared in TE buffer.

Bcr1 dsRNA hairpin sequence of one strand:

(SEQ ID NO: 93) AUGAAACUGCUGCAUGGCUUUCUGAUUAUUAUGCUGACCAUGCAUCUGAGCAUUCAGUAUGCGUAUGGCGGCCCGUUUCUGACCAAAUAUCUGUGCGAUCGCGUGUGCCAUAAACUGUGCGGCGAUGAAUUUGUGUGCAGCUGCAUUCAGUAUAAAAGCCUGAAAGGCCUGUGGUUUCCGCAUUGCCCGACCGGCAAAGCGAGCGUGGUGCUGCAUAACUUUCUGACCAGCCCGUUUUUUUUUUCGGGCUGGUCAGAAAGUUAUGCAGCACCACGCUCGCUUUGCCGGUCGGGCAAUGCGGAAACCACAGGCCUUUCAGGCUUUUAUACUGAAUGCAGCUGCACACAAAUUCAUCGCCGCACAGUUUAUGGCACACGCGAUCGCACAGAUAUUUGGUCAGAAACGGGCCGCCAUACGCAUACUGAAUGCUCAGAUGCAUGGUCAGCAUAAUAAUCAGAAAGCCAUGCAGCAGUUUCAU

Example 3: Treatment of Aphids (Rhopalosiphum maidis) with Bcr1 dsRNA

This Example demonstrates the ability to kill or decrease the fitness ofaphids, Rhopalosiphum maidis, through treatment with a dsRNA solution bytargeting expression of the Bcr1 gene (ACYPI32128), which is anessential gene for bacteriocyte regulation and function in insects.

Aphids are one of the most important agricultural insect pests. Theycause direct feeding damage to crops and serve as vectors of plantviruses. In addition, aphid honeydew promotes the growth of sooty moldand attracts nuisance ants. The use of chemical treatments,unfortunately still widespread, leads to the selection of resistantindividuals whose eradication becomes increasingly difficult.

Therapeutic Design:

dsRNA solutions are formulated with 0 (negative control), 0.5, 1, or 5μg/ml of Bcr1 dsRNA from Example 2 in 10 mL of TE buffer with 0.5%sucrose and essential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), fava bean plants are grown in a mixture of vermiculite andperlite and are infested with aphids. To limit maternal effects orhealth differences between plants, 5-10 adults from different plants aredistributed among 10 two-week-old plants, and allowed to multiply tohigh density for 5-7 days. For experiments, second and third instaraphids are collected from healthy plants and divided into treatments sothat each treatment receives approximately the same number ofindividuals from each of the collection plants.

Wells of a 96-well plate are filled with 200 μl of artificial aphid diet(Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) andthe plate is covered with parafilm to make a feeding sachet. Artificialdiet is either mixed with the solution of TE buffer (Tris HCl (1 mM)plus EDTA (0.1 mM) buffer, pH 7.2.) with 0.5% sucrose and essentialamino acids only as a negative control, or mixed with dsRNA solutionsdiluted in TE buffer containing varying concentrations of dsRNA. dsRNAsolutions are mixed with artificial diet to obtain final concentrationsbetween 0.5 to 5 μg/ml.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for 4 days. At the time that thesachet is replaced, aphids are also checked for mortality. An aphid iscounted as dead if it had turned brown or is at the bottom of the welland does not move during the observation. If an aphid is on the parafilmof the feeding sachet but not moving, it is assumed to be feeding andalive.

The survival rates of aphids treated with Bcr1 dsRNA are compared to theaphids treated with the negative control. The survival rate of aphidstreated with Bcr1 dsRNA is decreased as compared to the control treatedaphids.

Example 4: Production of Transgenic Grass Expressing Bcr1 dsRNAs

This Example demonstrates genetic modification and production of Bcr1dsRNA in a transgenic grass for delivery to aphids.

Transgenic forage grass blue grama, Bouteloua gracilis, that producesthe Bcr1 dsRNA molecules, through expression of a chimeric genestably-integrated into the plant genome, is produced by microprojectilebombardment, using a system based on the highly chlorophyllous andembryogenic cell line ‘TIANSJ98’ (Aguado-Santacruz et al., Theoreticaland Applied Genetics 104(5):763-771, 2002).

‘TIANSJ98’ cell line establishment and maintenance: The embryogenic,highly chlorophyllous ‘TIANSJ98’ cell line is obtained from culturingshoot apice-derived green calli in liquid MPC medium as described in(Aguado-Santacruz et al., Plant Cell Rep. 20:131-136, 2001). This cellline is subcultured every 20 days, transferring 1 ml of the cellsuspension into 24 ml of fresh MPC medium. The reasons for utilizing thefinely dispersed condition of the embryogenic calli are 1) tosynchronize the physiological stage of the target cells, 2) to maximizethe distribution of the totipotent material on the paper filters(optimizing the shoot cover of the bombarded plasmids), and 3) tofacilitate the identification of independent transformation events(green spots) within the dispersed cell clusters under selection.

Microprojectile Bombardment of Embryogenic Cells:

A binary transformation vector is used with the template fragment forBcr1 dsRNA expression. This plasmid contains the inverted repeat of thetarget Bcr1 gene under the control of a double 35S Cauliflower MosaicVirus promoter, and a leader sequence from Alfalfa Mosaic Virus(Aguado-Santacruz et al., Theoretical and Applied Genetics104(5):763-771, 2002).

The highly chlorophyllous embryogenic cell line ‘TIANSJ98’ is used asthe target for the microprojectile delivery experiments. The cells aredistributed onto 2.0-cm diameter paper-filter disks (approximately 2 gFW cells). Bombardment mixtures are as follows: 50 μL of M10 tungstenparticles (15 mg/ml), 10 μL of DNA (1 μg/ml), 50 μL of 2.5 M CaCl₂) and20 μL of 0.1 M espermidine are mixed in sequential order, vortexed for 5min and then briefly sonicated. The mixture is centrifuged at 10,000 rpmfor 10 s. 60 μL of the supernatant are removed and the rest is dispensedinto 5-μL aliquots for individual shoots. Bombardments are performedusing the Particle Inflow Gun (Finer et al., Plant Cell Rep. 11:323-328,1992). The particle/DNA mixture is placed in the center of the syringefilter unit. Embryogenic cells are covered with a 500-μm baffle, placedat a distance of 10 cm from the screen filter unit containing theparticles, and bombarded once in the vacuum chamber at 60 mmHg. Twodifferent osmotic media for pre- and post-bombardment treatments (0.4and 1 M mannitol supplied in solidified MPC medium) and threebombardment pressures (60, 80 and 100 PSI) are tested. Pre-bombardmenttreatment is applied 24 hr before shooting. After discharge, the paperfilters supporting the embryogenic cells are maintained for 3-days moreon the same osmotic medium used in the pre-bombardment treatment. Thus,a total of nine treatments are evaluated with ten dishes bombarded pertreatment. As a control, filters with suspension material are bombardedusing particles without DNA.

Selection of Stable Transformed Clones and Recovery of Plants:

After the 3-day post-bombardment osmotic treatment on MPC mediumcontaining 0.4 or 1 M manitol, but lacking antibiotic, the paper filterdisks supporting the bombarded cells are transferred onto MPC mediumcontaining 140 mg/l of kanamycin and incubated at 30±1° C. in whitelight provided by cold fluorescent lamps. The same procedure is followedfor cells bombarded but not subjected to osmotic treatment. Thekanamycin concentration is raised 2-months later to 150 or 160 mg/l. Thecells are sub cultured every 3 weeks and maintained for 8 months inselection. After this period, kanamycin-resistant clones are transferredto regeneration medium containing full-strength MS medium, 3% sucrose,2.5% phytagel (Sigma, St. Louis, Mo.) but no antibiotic. The regeneratedshoots are transferred for rooting to ½ MS containing 3.0 μM (0.56 mg/l)α-naphthaleneacetic acid, 2.5 μM (0.51 mg/l) indole-3-butyric acid and2.5% phytagel, and incubated at 30±1° C. under continuous fluorescentlight. Later, rooted plantlets are transferred to pots, hardened off,and grown to maturity in a greenhouse.

PCR Analysis for Transformation Verification:

Total genomic DNA is prepared from kanamycin-resistant and untransformedcontrol plants using the following protocol: Approximately 250 mg ofcells are collected in 2-ml Eppendorf tubes and ground to a fine powderin liquid N2 using a glass pestle attached to a homogenizer (Caframo,Stirrer type RZR). Powdered cells are re-suspended with 500 μL ofextraction buffer (7.0 M urea, 0.35 M NaCl, 0.05 M Tris-HCl, pH 8.0,0.02 M EDTA, 1% sarcosine) for at least 45 min. The cell homogenate isextracted with 1 vol of phenol/chloroform. The aqueous phase isseparated by centrifugation and then precipitated using an equal volumeof isopropyl alcohol. The precipitated DNA is washed once with 70%ethanol and resuspended in TE buffer (0.01 M Tris-HCl, 0.01 M EDTA, pH8.0).

For PCR analysis, 100 to 150 ng, are used for genomic DNAamplifications, in 25-μL reactions. Primers forward5′-aaactgctgcatggctttct-3′ (SEQ ID NO: 91) and reverse5′-acaggcctttcaggctttta-3′ (SEQ ID NO: 92) are designed for amplifyingan internal 169-bp fragment of the Bcr1 sense anti sense insertedfragment. PCR reactions are carried out using a Perkin Elmerthermocycler for 30 cycles. Reaction temperatures are denaturation 95°C. (2 min), annealing 56° C. (30 s), and extension 72° C. (30 s). The25-μL reaction volumes contain: 1 x PCR buffer, 0.25 mM of dNTPs, 2 mMMgCl2, 0.2 μM of primers and 2.5 u of Taq. The amplification productsare analyzed by electrophoreses in 1% agarose/SYBR green gels.

Example 5: Production of Transgenic Grass Producing colA Bacteriocin

This Example demonstrates genetic modification and production of thebacteriocyte regulatory peptide Coleoptericin A (colA) in a transgenicgrass for delivery to aphids.

Transgenic forage grass blue grama, Bouteloua gracilis, that producesColeoptericin A, through expression of a chimeric gene stably-integratedinto the plant genome, is produced by microprojectile bombardment, usinga system described in Example 4.

The embryogenic, highly chlorophyllous ‘TIANSJ98’ cell line issubcultured every 20 days, transferring 1 ml of the cell suspension into24 ml of fresh MPC medium.

Coleoptericin A (colA) (SEQ ID NO: 94)atgacccgcaccatgctgtttctggcgtgcgtggcggcgctgtatgtgtgcattagcgcgaccgcgggcaaaccggaagaatttgcgaaactgagcgatgaagcgccgagcaacgatcaggcgatgtatgaaagcattcagcgctatcgccgctttgtggatggcaaccgctataacggcggccagcagcagcagcagcagccgaaacagtgggaagtgcgcccggatctgagccgcgatcagcgcggcaacaccaaagcgcaggtggaaattaacaaaaaaggcgataaccatgatattaacgcgggctggggcaaaaacattaacggcccggatagccataaagatacctggcatgtgggcggcagcgtgcgctgg

A transformation plasmid is constructed for Coleoptericin A (colA)expression. The plasmid contains the nucleic acid for colA under thecontrol of a double 35S Cauliflower Mosaic Virus promoter, and a leadersequence from Alfalfa Mosaic Virus (Aguado-Santacruz et al., Theoreticaland Applied Genetics 104(5):763-771, 2002).

The highly chlorophyllous embryogenic cell line ‘TIANSJ98’ is used asthe target for the microprojectile delivery experiments. The cells aredistributed onto 2.0-cm diameter paper-filter disks (approximately 2 gFW cells). Bombardment mixtures are as follows: 50 μL of M10 tungstenparticles (15 mg/ml), 10 μL of DNA (1 μg/ml), 50 μL of 2.5 M CaCl₂) and20 μL of 0.1 M espermidine are mixed in sequential order, vortexed for 5min and then briefly sonicated. The mixture is centrifuged at 10,000 rpmfor 10 s. 60 μL of the supernatant are removed and the rest is dispensedinto 5-μL aliquots for individual shoots. Bombardments are performedusing the Particle Inflow Gun (Finer et al., Plant Cell Rep. 11:323-328,1992). The particle/DNA mixture is placed in the center of the syringefilter unit. Embryogenic cells are covered with a 500-μm baffle, placedat a distance of 10 cm from the screen filter unit containing theparticles, and bombarded once in the vacuum chamber at 60 mmHg. Twodifferent osmotic media for pre- and post-bombardment treatments (0.4and 1 M mannitol supplied in solidified MPC medium) and threebombardment pressures (60, 80 and 100 PSI) are tested. Pre-bombardmenttreatment is applied 24 hr before shooting. After discharge, the paperfilters supporting the embryogenic cells are maintained for 3-days moreon the same osmotic medium used in the pre-bombardment treatment. Thus,a total of nine treatments are evaluated with ten dishes bombarded pertreatment. As a control, filters with suspension material are bombardedusing particles without DNA.

Selection of Stable Transformed Clones and Recovery of Plants:

After the 3-day post-bombardment osmotic treatment on MPC mediumcontaining 0.4 or 1 M manitol, but lacking antibiotic, the paper filterdisks supporting the bombarded cells are transferred onto MPC mediumcontaining 140 mg/l of kanamycin and incubated at 30±1° C. in whitelight provided by cold fluorescent lamps. The same procedure is followedfor cells bombarded but not subjected to osmotic treatment. Thekanamycin concentration is raised 2-months later to 150 or 160 mg/l. Thecells are sub cultured every 3 weeks and maintained for 8 months inselection. After this period, kanamycin-resistant clones are transferredto regeneration medium containing full-strength MS medium, 3% sucrose,2.5% phytagel (Sigma, St. Louis, Mo.) but no antibiotic. The regeneratedshoots are transferred for rooting to ½ MS containing 3.0 μM (0.56 mg/l)α-naphthaleneacetic acid, 2.5 μM (0.51 mg/l) indole-3-butyric acid and2.5% phytagel, and incubated at 30±1° C. under continuous fluorescentlight. Later, rooted plantlets are transferred to pots, hardened off,and grown to maturity in a greenhouse.

PCR Analysis for Transformation Verification:

Total genomic DNA is prepared from kanamycin-resistant and untransformedcontrol plants using the following protocol: Approximately 250 mg ofcells are collected in 2-ml Eppendorf tubes and ground to a fine powderin liquid N2 using a glass pestle attached to a homogenizer (Caframo,Stirrer type RZR). Powdered cells are re-suspended with 500 μL ofextraction buffer (7.0 M urea, 0.35 M NaCl, 0.05 M Tris-HCl, pH 8.0,0.02 M EDTA, 1% sarcosine) for at least 45 min. The cell homogenate isextracted with 1 vol of phenol/chloroform. The aqueous phase isseparated by centrifugation and then precipitated using an equal volumeof isopropyl alcohol. The precipitated DNA is washed once with 70%ethanol and resuspended in TE buffer (0.01 M Tris-HCl, 0.01 M EDTA, pH8.0).

For PCR analysis, 100 to 150 ng, are used for genomic DNAamplifications, in 25-μL reactions. Primers forward5′-caacgatcaggcgatgtatg-3′ (SEQ ID NO: 95) and reverse5′-ttaatttccacctgcgcttt-3′ (SEQ ID NO: 96) are designed for amplifyingan internal 165-bp fragment of the colA gene inserted fragment. PCRreactions are carried out using a Perkin Elmer thermocycler for 30cycles. Reaction temperatures are denaturation 95° C. (2 min), annealing56° C. (30 s), and extension 72° C. (30 s). The 25-μL reaction volumescontain: 1×PCR buffer, 0.25 mM of dNTPs, 2 mM MgCl2, 0.2 μM of primersand 2.5 u of Taq. The amplification products are analyzed byelectrophoreses in 1% agarose/SYBR green gels.

Example 6: Production of Transgenic Grass Producing colA Bacteriocin

This example demonstrates genetic modification and production of colAbacteriocin in a transgenic grass for delivery to aphids.

Transgenic forage grass blue grama, Bouteloua gracilis, that producescolA bacteriocin, through expression of a chimeric genestably-integrated into the plant genome, is produced by microprojectilebombardment, using a system based on the highly chlorophyllous andembryogenic cell line TIANSJ98′ (Aguado-Santacruz et al., 2002.Theoretical and Applied Genetics, 104(5), 763-771).

‘TIANSJ98’ cell line establishment and maintenance: The embryogenic,highly chlorophyllous ‘TIANSJ98’ cell line is obtained from culturingshoot apice-derived green calli in liquid MPC medium as described in(Aguado-Santacruz et al., 2001. ex Steud. Plant Cell Rep 20:131-136).This cell line is subcultured every 20 days, transferring 1 ml of thecell suspension into 24 ml of fresh MPC medium. The reasons forutilizing the finely dispersed condition of the embryogenic calli are 1)to synchronize the physiological stage of the target cells, 2) tomaximize the distribution of the totipotent material on the paperfilters (optimizing the shoot cover of the bombarded plasmids), and 3)to facilitate the identification of independent transformation events(green spots) within the dispersed cell clusters under selection.

Microprojectile Bombardment of Embryogenic Cells

A transformation vector is used with the template fragment for colAbacteriocin expression.

colA bacteriocin (SEQ ID NO: 94)atgacccgcaccatgctgtttctggcgtgcgtggcggcgctgtatgtgtgcattagcgcgaccgcgggcaaaccggaagaatttgcgaaactgagcgatgaagcgccgagcaacgatcaggcgatgtatgaaagcattcagcgctatcgccgctttgtggatggcaaccgctataacggcggccagcagcagcagcagcagccgaaacagtgggaagtgcgcccggatctgagccgcgatcagcgcggcaacaccaaagcgcaggtggaaattaacaaaaaaggcgataaccatgatattaacgcgggctggggcaaaaacattaacggcccggatagccataaagatacctggcatgtgggcggcagcgtgcgctgg

This plasmid contains the nucleic acid for colA bacteriocin under thecontrol of a double 35S Cauliflower Mosaic Virus promoter, and a leadersequence from Alfalfa Mosaic Virus (Aguado-Santacruz et al., 2002.Theoretical and Applied Genetics, 104(5), 763-771).

The highly chlorophyllous embryogenic cell line ‘TIANSJ98’ is used asthe target for the microprojectile delivery experiments. The cells aredistributed onto 2.0-cm diameter paper-filter disks (approximately 2 gFW cells). Bombardment mixtures are as follows: 50 μL of M10 tungstenparticles (15 mg/ml), 10 μL of DNA (1 μg/ml), 50 μL of 2.5 M CaCl₂) and20 μL of 0.1 M espermidine are mixed in sequential order, vortexed for 5min and then briefly sonicated. The mixture is centrifuged at 10,000 rpmfor 10 s. 60 μL of the supernatant are removed and the rest is dispensedinto 5-μL aliquots for individual shoots.

Bombardments are performed using the Particle Inflow Gun (Finer et al.,1992. Plant Cell Rep 11:323-328). The particle/DNA mixture is placed inthe center of the syringe filter unit. Embryogenic cells are coveredwith a 500-μm baffle, placed at a distance of 10 cm from the screenfilter unit containing the particles, and bombarded once in the vacuumchamber at 60 mmHg. Two different osmotic media for pre- andpost-bombardment treatments (0.4 and 1 M mannitol supplied in solidifiedMPC medium) and three bombardment pressures (60, 80 and 100 PSI) aretested. Pre-bombardment treatment is applied 24 hr before shooting.

After discharge, the paper filters supporting the embryogenic cells aremaintained for 3-days more on the same osmotic medium used in thepre-bombardment treatment. Thus, a total of nine treatments areevaluated with ten dishes bombarded per treatment. As a control, filterswith suspension material are bombarded using particles without DNA.

Selection of Stable Transformed Clones and Recovery of Plants

After the 3-day post-bombardment osmotic treatment on MPC medium with0.4 or 1 M manitol, but lacking antibiotic, the paper filter diskssupporting the bombarded cells are transferred onto MPC mediumcontaining 140 mg/l of kanamycin and incubated at 30±1° C. in whitelight provided by cold fluorescent lamps. The same procedure is followedfor cells bombarded but not subjected to osmotic treatment. Thekanamycin concentration is raised 2-months later to 150 or 160 mg/l. Thecells are subcultured every 3 weeks and maintained for 8 months inselection. After this period, kanamycin-resistant clones are transferredto regeneration medium with full-strength MS medium, 3% sucrose, 2.5%phytagel (Sigma, St. Louis, Mo.) but no antibiotic. The regeneratedshoots are transferred for rooting to ½ MS with 3.0 μM (0.56 mg/l)α-naphthaleneacetic acid, 2.5 μM (0.51 mg/l) indole-3-butyric acid and2.5% phytagel, and incubated at 30±1° C. under continuous fluorescentlight. Later, rooted plantlets are transferred to pots, hardened off,and grown to maturity in a greenhouse.

PCR Analysis for Transformation Verification

Total genomic DNA is prepared from kanamycin-resistant and untransformedcontrol plants using the following protocol: Approximately 250 mg ofcells are collected in 2-ml Eppendorf tubes and ground to a fine powderin liquid N2 using a glass pestle attached to a homogenizer (Caframo,Stirrer type RZR). Powdered cells are re-suspended with 500 μL ofextraction buffer (7.0 M urea, 0.35 M NaCl, 0.05 M Tris-HCl, pH 8.0,0.02 M EDTA, 1% sarcosine) for at least 45 min. The cell homogenate isextracted with 1 vol of phenol/chloroform. The aqueous phase isseparated by centrifugation and then precipitated using an equal volumeof isopropyl alcohol. The precipitated DNA is washed once with 70%ethanol and resuspended in TE buffer (0.01 M Tris-HCl, 0.01 M EDTA, pH8.0).

For PCR analysis, 100 to 150 ng, are used for genomic DNAamplifications, in 25-μL reactions. Primers forward5′-caacgatcaggcgatgtatg-3′ and reverse 5′-ttaatttccacctgcgcttt-3′ aredesigned for amplifying an internal 165-bp fragment of the ColA geneinserted fragment. PCR reactions are carried out using a Perkin Elmerthermocycler for 30 cycles. Reaction temperatures are denaturation 95°C. (2 min), annealing 56° C. (30 s), and extension 72° C. (30 s). The25-μL reaction volumes contain: 1×PCR buffer, 0.25 mM of dNTPs, 2 mMMgCl2, 0.2 μM of primers and 2.5 u of Taq. The amplification productsare analyzed by electrophoreses in 1% agarose/SYBR green gels.

Example 7: Production of a cDNA Library from Green Peach Aphid (Myzuspersicae)

This Example demonstrates the production of a cDNA library from greenpeach aphid larvae (Myzus persicae).

Total RNA Isolation from Aphid Larvae and cDNA Library Preparation:

Total RNA from 0.9 g of whole first-instar larvae (4 to 5 dayspost-hatch; held at 16° C.) is extracted using the following phenol/TRIREAGENT®-based method (MOLECULAR RESEARCH CENTER, Cincinnati, Ohio).Larvae are homogenized at room temperature in a 15 mL homogenizer with10 mL of TRI REAGENT® until a homogenous suspension is obtained.Following 5 min. incubation at room temperature, the homogenate isdispensed into 1.5 mL microfuge tubes (1 mL per tube), 200 μL ofchloroform is added, and the mixture is vigorously shaken for 15seconds. After allowing the extraction to sit at room temperature for 10min, the phases are separated by centrifugation at 12,000×g at 4° C. Theupper phase (including about 0.6 mL) is carefully transferred intoanother sterile 1.5 mL tube, and an equal volume of room temperatureisopropanol is added. After incubation at room temperature for 5 to 10min, the mixture is centrifuged 8 min at 12,000×g (4° C. or 25° C.).

The supernatant is carefully removed and discarded, and the RNA pelletis washed twice by vortexing with 75% ethanol, with recovery bycentrifugation for 5 min at 7,500×g (4° C. or 25° C.) after each wash.The ethanol is carefully removed, the pellet is allowed to air-dry for 3to 5 min, and then is dissolved in nuclease-free sterile water. The RNAconcentration is determined by measuring the absorbance (A) at 260 nmand 280 nm. The RNA is stored at −80° C., and the RNA quality isdetermined by running an aliquot through a 1% agarose gel.

The larval total RNA is converted into a cDNA library using randompriming. The larval cDNA library is sequenced at ½ plate scale by GS FLX454 Titanium™ series chemistry at EUROFINS MWG Operon, which results inover 600,000 reads with an average read length of 348 bp. 350,000 readsare assembled into over 50,000 contigs. Both the unassembled reads andthe contigs are converted into BLASTable databases using the publiclyavailable program, FORMATDB (available from NCBI).

Example 8: Production of Bcr1 dsRNA

This Example demonstrates the production and purification of a syntheticdsRNA from a cDNA library.

Experimental Design:

Bcr1 gene (ACYP132128) is an essential gene for bacteriocyte regulationand function in insects. The cDNA described in the Example 6 is used forBcr1 dsRNA synthesis prepared by PCR using the primer pairs: Forward5′-aaactgctgcatggctttct-3′ (SEQ ID NO: 90) and reverse5′-acaggcctttcaggctttta-3′ (SEQ ID NO: 91). For the target gene region,two separate PCR amplifications are performed. The first PCRamplification introduces a T7 promoter sequence at the 5′ end((TTAATACGACTCACTATAGGGAGA; SEQ ID NO: 92) of the amplified sensestrands. The second reaction incorporates the T7 promoter sequence atthe 5′ ends of the antisense strands. The two PCR amplified fragmentsfor each region of the target gene Bcr1 are then mixed in equal amounts,and the mixture is used as a transcription template for dsRNAproduction. Double-stranded RNA for insect bioassay is synthesized andpurified using an AMBION® MEGASCRIPT® RNAi kit following themanufacturer's instructions (INVITROGEN) or HiScribe® T7 In VitroTranscription Kit following the manufacturer's instructions (New EnglandBiolabs, Ipswich, Mass.). The concentration of dsRNAs against Bcr1 ismeasured using a NANODROP™ 8000 spectrophotometer (THERMO SCIENTIFIC,Wilmington, Del.) and the purified dsRNA molecules are prepared in TEbuffer.

Bcr1 dsRNA hairpin sequence of one strand:

(SEQ ID NO: 93) AUGAAACUGCUGCAUGGCUUUCUGAUUAUUAUGCUGACCAUGCAUCUGAGCAUUCAGUAUGCGUAUGGCGGCCCGUUUCUGACCAAAUAUCUGUGCGAUCGCGUGUGCCAUAAACUGUGCGGCGAUGAAUUUGUGUGCAGCUGCAUUCAGUAUAAAAGCCUGAAAGGCCUGUGGUUUCCGCAUUGCCCGACCGGCAAAGCGAGCGUGGUGCUGCAUAACUUUCUGACCAGCCCGUUUUUUUUUUCGGGCUGGUCAGAAAGUUAUGCAGCACCACGCUCGCUUUGCCGGUCGGGCAAUGCGGAAACCACAGGCCUUUCAGGCUUUUAUACUGAAUGCAGCUGCACACAAAUUCAUCGCCGCACAGUUUAUGGCACACGCGAUCGCACAGAUAUUUGGUCAGAAACGGGCCGCCAUACGCAUACUGAAUGCUCAGAUGCAUGGUCAGCAUAAUAAUCAGAAAGCCAUGCAGCAGUUUCAU

Example 9: Treatment of Aphids (Myzus persicae) with a Solution of Bcr1dsRNA

This Example demonstrates the ability to kill or decrease the fitness ofaphids, Myzus persicae, by treating them with a dsRNA solution bytargeting expression of the Bcr1 gene (ACYP132128), which is anessential gene for bacteriocyte regulation and function in insects.

Aphids are one of the most important agricultural insect pests. Theycause direct feeding damage to crops and serve as vectors of plantviruses. In addition, aphid honeydew promotes the growth of sooty moldand attracts nuisance ants. The use of chemical treatments,unfortunately still widespread, leads to the selection of resistantindividuals whose eradication becomes increasingly difficult.

Therapeutic Design:

dsRNA solutions are formulated with 0 (negative control), 0.5, 1, or 5μg/mL of Bcr1 dsRNA from Example 7 in 10 mL of TE buffer with 0.5%sucrose and essential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 hr light photoperiod; 60±5% RH;20±2° C.), plants are grown in a mixture of vermiculite and perlite andare infested with aphids. To limit maternal effects or healthdifferences between plants, 5-10 adults from different plants aredistributed among 10 two-week-old plants, and allowed to multiply tohigh density for 5-7 days. For experiments, second and third instaraphids are collected from healthy plants and divided into treatments sothat each treatment receives approximately the same number ofindividuals from each of the collection plants.

Wells of a 96-well plate are filled with 200 μl of artificial aphid diet(Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) andthe plate is covered with parafilm to make a feeding sachet. Artificialdiet is either mixed with the solution of TE buffer (Tris HCl (1 mM)plus EDTA (0.1 mM) buffer, pH 7.2.) with 0.5% sucrose and essentialamino acids only as a negative control, or mixed with dsRNA solutionsdiluted in TE buffer containing varying concentrations of dsRNA. dsRNAsolutions are mixed with artificial diet to obtain final concentrationsbetween 0.5 to 5 μg/ml.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for 4 days. At the time that thesachet is replaced, aphids are also checked for mortality. An aphid iscounted as dead if it had turned brown or is at the bottom of the welland does not move during the observation. If an aphid is on the parafilmof the feeding sachet but not moving, it is assumed to be feeding andalive.

The survival rates of aphids treated with Bcr1 dsRNA are compared to theaphids treated with the negative control. The survival rate of aphidstreated with Bcr1 dsRNA is decreased as compared to the control treatedaphids.

Example 10: Topical-Plant Delivery of Bacteriocyte Specific dsRNA forCrop Protection

This Example demonstrates the ability to deliver bacteriocyte specificdsRNA by topical application on tobacco plant leaves. dsRNA targets theexpression of the Bcr1 gene (ACYP132128), which is an essential gene forbacteriocyte regulation and function in insects.

Therapeutic Design:

dsRNA-LDH spray solutions are formulated with Bcr1 dsRNA produced inExample 7 at 1:0 (negative control), 1:1, 1:2, or 1:3 ratio of dsRNA:LDHat 125 uL/cm² of leave surface.

Preparation and Characterization of LDH Nanosheets:

Sheet-like clay nanoparticles, specifically positively charged LDH, areexcellent nanocarrier systems for dsRNA, to deliver RNAi as stable sprayformulations for crop protection (Mitter et al., Nature Plants 1-10,2017). LDH conjugates strongly adhere to the leaf surface even aftervigorous rinsing, and enhance the stability of dsRNA for a longer periodunder environmental conditions. The sustained release of dsRNA isfacilitated through the formation of carbonic acid on the leaf surfacefrom atmospheric CO₂ and humidity, which helps degrade the LDHnanosheets.

LDH nanosheets are prepared according to (Mitter et al., Nature Plants1-10, 2017) by modified non-aqueous precipitation, followed by heattreatment, purification and dispersion in water to get an averageparticle size of 45 nm. The particles (five repeated LDH samples) arecharacterized by the Nanosizer Nano ZS instrument (Malvern Instruments)to obtain the Z average size and Pdl and imaged using JEOL JSM-2010TEM22. The chemical composition and crystal structure are verified bypowder XRD with five LDH samples and Fourier transform infraredspectroscopy with attenuated total reflection mode, using a RigakuMiniflex X-Ray diffractometer and a Nicolet 6700 FT-IR (Thermo ElectronCorporation), respectively.

dsRNA Loading on LDH:

To define optimal and complete loading of the dsRNA hairpin constructobtained in Example 7 into LDH nanosheets, the ratio of in vitrotranscribed dsRNA (500 ng) to LDH (dsRNA-LDH (w/w)) is assayed at 1:1,1:2, 1:3, 1:4, 1:5 and 1:10 multiple times. dsRNA and LDH are incubatedin a total volume of 10 μL at room temperature for 30 min with gentleorbital agitation. Complete dsRNA loading is assessed by retention ofdsRNA-LDH complexes in the well of a 1% agarose gel. Appropriate loadingratios are consistent, irrespective of the scale-up volume required.

Release of dsRNA from dsRNA-LDH complexes and stability of the releaseddsRNA are tested as described in (Mitter et al., Nature Plants 1-10,2017).

Northern Blot Analysis for dsRNA Detection within the Leaves:

To detect dsRNA uptake, N. tabacum plants (three replicates) are sprayedwith either LDH, Bcr1-dsRNA or Bcr1-dsRNA-LDH at day 0. The ratios ofthe complex tested are: 1:1, 1:2, or 1:3 ratios of dsRNA-LDH. Plants aregrown in UQ23 soil in 10 cm wide pots under glasshouse conditions(average temperature 25° C. with natural light). The apex of theseplants is covered using a masking tape at the time of the spray. Newleaves that emerge 20 days after the spray are collected. Total RNA isextracted by TRIzol extraction and enriched for small RNAs (Mitter etal., Am. Phytopathological Soc. 16:936-944, 2003). Small RNAs (20 μg)for each treatment are run on a 15% (wt/vol) denaturing ureapolyacrylamide gel (PAGE). ZR Small-RNA ladder (Zymo Research) labelledwith DIG at the 3′ end is used as a marker. The blots are transferred bytrans-blot SD semi-dry transfer unit (Bio-Rad) on a Hybond-N membrane(Roche). Blots are processed as per manufacture's recommendation usingDIG-labelled Bcr1 24 nt probe (5-atgctgaccatgcatctgagcatt), proprietarybuffer set (Roche). Following hybridization, filters are detected usingthe CSPD chemiluminescent alkaline phosphatase substrate. Thequantification analysis is performed using the NIH Image 1.6 software.

Example 11: Solid Phase Synthesis of a PNA

This Example demonstrates solid phase synthesis of a PNA.

Therapeutic Design:

complementary antisense PNA constructs against the bacteriocyte targetgene Bcr1: gaatgcagctgc

Experimental Design:

The PNA antisense is synthesized automatically (MilliGen 9050 peptidesynthesizer) by the solid-phase method using standard Fmoc(N-(9-fluorenyl)methoxycarbonyl) chemistry in a continuous flow mode.

PNA antisense purification is performed by reversed-phasehigh-performance liquid chromatography (RP-HPLC) with UV detection at260 nm using a semi-prep column C18 (10 μm, 300×7.7 mm, Xterra Waters,300 Å), eluting with water containing 0.1% TFA (eluent A) andacetonitrile containing 0.1% TFA (eluent B); elution gradient: from 100%A to 50% B in 30 min, flow: 4 ml/min. The purity and identity of thepurified PNA antisense is examined by ultra-performance liquidchromatography tandem mass-spectrometry (UPLC-MS; Waters Acquityequipped with ESI-Q analizer) using an Acquity UPLC BEH C18; 2.1×50 MM,1.7 μm column. The expected mass peaks are observed for the correctamino and nucleic acid sequence.

Example 12: Production of Cy3 Labeled PNA

This Example demonstrates joining the PNA described in Example 10 to Cy3dye as a marker to tag the PNA through click chemistry.

Therapeutic Design:

PNA with dibenzylcyclooctyne (DBCO) modification and Cy3 dye with azidemodification: Cy3-gaatgcagctgc.

Experimental Design:

To prepare for the click reaction the PNA synthesized in Example 10 islabeled with DBCO (Glen Research, Sterling, Va.). DBCO-sulfo-NHS esteris dissolved at a concentration of 5.2 mg per 60 μL in water oranhydrous DMSO. This stock solution is used to conjugate theamino-modified PNA in sodium carbonate/bicarbonate conjugation buffer,pH=˜9.

For a 0.2 μmol synthesis of PNA, PNA is dissolved in 500 μL ofconjugation buffer. Approx. a 6 fold excess (6 μL) of DBCO-sulfo-NHSester solution is added to the dissolved PNA. The mixture is vortexedand incubated at room temperature for 2-4 hours up to about overnight.The conjugated PNA is desalted on a desalting column (Glen Research,Sterling, Va.) to remove salts and organics.

Cy3-azide modified dye is obtained from Sigma Aldrich (777315). For theclick reaction, 1 mg of Cy3-azide is dissolved in 150 μL of DMSO. TheCy3-azide solution is added to 10 OD of DBCO conjugated PNA in 100 μL ofwater. The mixture is incubated at room temperature overnight. Theligated PNAs are desalted on a desalting column (Glen Research,Sterling, Va.) to remove salts and organics.

Example 13: Treatment of Aphids (Myzus persicae) with a Solution ofCy3-PNA

This Example demonstrates the ability to kill or decrease the fitness ofaphids, Myzus persicae, by treating them with a Cy3-PNA constructs thattargets expression of the Bcr1 gene (ACYP132128), which is an essentialgene for bacteriocyte regulation and function in insects.

Aphids are one of the most important agricultural insect pests. Theycause direct feeding damage to crops and serve as vectors of plantviruses. In addition, aphid honeydew promotes the growth of sooty moldand attracts nuisance ants. The use of chemical treatments,unfortunately still widespread, leads to the selection of resistantindividuals whose eradication becomes increasingly difficult.

Therapeutic Design:

Cy3-PNA solutions are formulated with 0, 0.5, 1, 5 or 10 mg/L of Cy3-PNAfrom Example 11 in 10 mL of 0.5% sucrose and essential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), plants are grown in a mixture of vermiculite and perlite andare infested with aphids. To limit maternal effects or healthdifferences between plants, 5-10 adults from different plants aredistributed among 10 two-week-old plants, and allowed to multiply tohigh density for 5-7 days. For experiments, second and third instaraphids are collected from healthy plants and divided into treatments sothat each treatment receives approximately the same number ofindividuals from each of the collection plants.

Wells of a 96-well plate are filled with 200 μl of artificial aphid diet(Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) andthe plate is covered with parafilm to make a feeding sachet. Artificialdiet is either mixed with the solution of sterile water with 0.5%sucrose and essential amino acids only as a negative control, or mixedwith Cy3-PNA solutions. Cy3-PNA solutions are mixed with artificial dietto obtain final concentrations between 0.5 to 10 mg/L.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for 4 days. At the time that thesachet is replaced, aphids are also checked for mortality. An aphid iscounted as dead if it had turned brown or is at the bottom of the welland does not move during the observation. If an aphid is on the parafilmof the feeding sachet but not moving, it is assumed to be feeding andalive.

The survival rates of aphids treated with Bcr1 specific Cy3-PNA arecompared to the aphids treated with the negative control. The survivalrate of aphids treated with Bcr1 specific Cy3-PNA is decreased ascompared to the control treated aphids.

Example 14: Topical-Plant Delivery of Cy3-PNA for Crop Protection

This Example demonstrates the ability to deliver Cy3-PNA by topicalapplication on tobacco plant leaves. Cy3-PNA targets the expression ofthe Bcr1 gene (ACYP132128), which is an essential gene for bacteriocyteregulation and function in insects.

Therapeutic Design:

Cy3-PNA-LDH spray solutions are formulated with Cy3-PNA synthesized inExample 11 at 1:0 (negative control), 1:1, 1:2, or 1:3 ratio ofCy3-PNA:LDH at 125 uL/cm² of leave surface.

Preparation and Characterization of LDH Nanosheets:

Sheet-like clay nanoparticles, specifically positively charged LDH, areexcellent nanocarrier systems to deliver nucleic acids as stable sprayformulations for crop protection (Mitter et al., Nature Plants 1-10,2017). LDH conjugates strongly adhere to the leaf surface even aftervigorous rinsing, and enhance the stability of PNAs for a longer periodunder environmental conditions. The sustained release of PNAs isfacilitated through the formation of carbonic acid on the leaf surfacefrom atmospheric CO₂ and humidity, which helps degrade the LDHnanosheets.

LDH nanosheets are prepared according to (Mitter et al., Nature Plants1-10, 2017) by modified non-aqueous precipitation, followed by heattreatment, purification, and dispersion in water to get an averageparticle size of 45 nm. The particles (five repeated LDH samples) arecharacterized by the Nanosizer Nano ZS instrument (Malvern Instruments)to obtain the Z average size and Pdl and imaged using JEOL JSM-2010TEM22. The chemical composition and crystal structure are verified bypowder XRD with five LDH samples and Fourier transform infraredspectroscopy with attenuated total reflection mode, using a RigakuMiniflex X-Ray diffractometer and a Nicolet 6700 FT-IR (Thermo ElectronCorporation), respectively.

Cy3-PNA Loading on LDH:

To define optimal and complete loading of the Cy3-PNAs obtained inExample 11 into LDH nanosheets, the ratio of Cy3-PNA (0.5 μg) to LDH(Cy3-PNA-LDH (w/w)) is assayed at 1:1, 1:2, 1:3, 1:4, 1:5 and 1:10multiple times. Cy3-PNA and LDH are incubated in a total volume of 10 μLat room temperature for 30 min with gentle orbital agitation. CompleteCy3-PNA loading is assessed by retention of Cy3-PNA-LDH complexes in awell of a 1% agarose gel. Appropriate loading ratios are consistent,irrespective of the scale-up volume required.

Confocal Imaging for Cy3-PNA Detection within Plant Leaves:

Surface-sterilized seeds of Arabidopsis thaliana Col-0 are vernalized,plated and vertically grown at 21° C. (16 h/8 h day/night) for 11 dayson solid MS media.

To detect uptake of Cy3-PNA, 11-day-old A. thaliana seedlings inreplicates of three are treated with 3 μL droplets of Cy3 only, Cy3-PNA(1 μg) and Cy3-PNA-LDH (1:3). Droplets are applied onto individualleaves. After 48 h the leaves are rinsed twice for 2 min in 3 ml ofwater with vigorous pipetting and visualized below the epidermis asflorescence remained on the surface in case of LDH treatments. Naturalchlorophyll florescence is detected with 650-800 nm bandpath filter.Z-stacks are used to verify that the florescence observed in the spongymesophyll and xylem are internalized. The leaves are viewed under aZeiss LSM510 META (Carl Zeiss) confocal laser scanning microscope. Cy3florescence is visualized by excitation with a HeNe laser at 543 nm anddetected with a 560-615 nm bandpath filter.

Example 15: Treatment of Aphids with a Solution that Increases BodyTemperature

This Example demonstrates the ability to kill or decrease the fitness ofaphids by treating them with prostaglandin. Prostaglandins areeicosanoids associated with immune function that rapidly induce fever invertebrates and invertebrates. This example demonstrates that the effectof prostaglandin on aphids is mediated through the modulation ofbacterial populations endogenous to the aphid that are sensitive to anincrease in temperature generated by prostaglandin. One targetedbacterial strain is Buchnera.

Therapeutic Design:

The prostaglandin solution E2 (PGE2) was formulated with 0 (negativecontrol), 10, 20 or 50 μg/μl of prostaglandin formulated in a solutionof ethanol and sterile water with 0.5% sucrose and essential aminoacids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), fava bean plants are grown in a mixture of vermiculite andperlite at 24° C. with 16 h of light and 8 h of darkness. To limitmaternal effects or health differences between plants, 5-10 adults fromdifferent plants are distributed among 10 two-week-old plants, andallowed to multiply to high density for 5-7 days. For experiments,second and third instar aphids are collected from healthy plants anddivided into treatments so that each treatment receives approximatelythe same number of individuals from each of the collection plants.

Prostaglandin solutions are made by dissolving prostaglandin(SIGMA-ALDRICH, P5640) in sterile water and ethanol (1:1) with 0.5%sucrose and essential aminoacids. Wells of a 96-well plate are filledwith 200 μl of artificial aphid diet (Febvay, et al., Canadian Journalof Zoology, 1988, 66(11): 2449-2453) and the plate is covered withparafilm to make a feeding sachet. Artificial diet is either mixed withsterile water and with 0.5% sucrose and essential aminoacids as anegative control or a prostaglandin solution containing one of theconcentrations of prostaglandin. Prostaglandin solutions are mixed withartificial diet to get final concentrations between 10 and 50 μg/μL.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for four days. At the time thatthe sachet is replaced, aphids are also checked for mortality. An aphidis counted as dead if it had turned brown or is at the bottom of thewell and does not move during the observation. If an aphid is on theparafilm of the feeding sachet but not moving, it is assumed to befeeding and alive.

The status of Buchnera in aphid samples is assessed by PCR. Total DNA isisolated from control (non-prostaglandin treated) and prostaglandintreated individuals using an Insect DNA Kit (OMEGA, Bio-tek) accordingto the manufacturer's protocol. The primers for Buchnera, forward primer5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 97) and reverse primer5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 98), are designed based on 23S-5SrRNA sequences obtained from the Buchnera genome (Accession Number: GCA000009605.1) (Shigenobu, et al., Nature 200.407, 81-86) using Primer 5.0software (Primer-E Ltd., Plymouth, UK). The PCR amplification cyclesincluded an initial denaturation step at 95° C. for 5 min, 35 cycles of95° C. for 30 s, 55° C. for 30 s, and 72° C. for 60 s, and a finalextension step of 10 min at 72° C. Amplification products fromprostaglandin treated and control samples are analyzed on 1% agarosegels, stained with SYBR safe, and visualized using an imaging System.Prostaglandin treated aphids show a reduction of Buchnera specificgenes.

The survival rates of aphids treated with prostaglandin solution arecompared to the aphids treated with the negative control. The survivalrate of aphids treated with prostaglandin solution is decreased comparedto the control.

Example 16: Treatment of Aphids with dsRNA to Stimulate an Insect ImmuneResponse to Decrease Fitness

This Example demonstrates that the treatment of aphids withdouble-stranded RNA (dsRNA) resulted in the knock-down ofimmunoregulatory genes to induce an immune response and decrease aphidfitness. By inducing an immune response by inhibiting animmunoregulatory gene, specifically, Cact a negative regulator of theToll pathway, which is the primary immunity pathway in aphids, to induceToll pathway activation to express and secrete antimicrobial peptides,lysozymes, and prophenoloxidase that ultimately affect bacterialpopulations endogenous to the aphid. This Example demonstrates that theeffect of lowering the levels of Cact in aphids to upregulate thesystemic immune responses leading to the dysregulation of bacterialpopulations endogenous to the aphid that are sensitive to the increasein Aphid immune responses generated by Toll pathway activation. Onetargeted bacterial strain is Buchnera.

Therapeutic Design:

5^(th) instar LSR-1 aphids are microinjected. The injection solutionswill be either dd-water (negative control) or dsRNA diluted in dd-waterat various concentrations (8 or 60 ng/aphid; see below).

Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), A. pisum will be grown onfava bean plants (Vroma vicia faba from Johnny's Selected Seeds) in aclimate-controlled incubator (16 h light/8 h dark photoperiod; 60±5% RH;25±2° C.). Prior to being used for aphid rearing, fava bean plants willbe grown in potting soil at 24° C. with 16 h of light and 8 h ofdarkness. To limit maternal effects or health differences betweenplants, 5-10 adults from different plants will be distributed among 10two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, fifth instar aphids will be collected fromhealthy plants and divided into 2 different treatment groups: 1) water(negative control) or 2) dsRNA against ApGLNT1 (at the concentrationsindicated herein).

Microinjection Delivery Experimental Design

Microinjection will be performed using NanoJet III Auto-NanoliterInjector with an in-house pulled borosilicate needle (DrummondScientific; Cat#3-000-203-G/XL). Aphids will be transferred using apaint brush to a tubing system connected to vacuum which held the aphidin place during the microinjection. The injection site will be at theventral thorax of the aphid. The injection volume will be 20 nl foradult aphids at a rate of 2 nl/sec. Each treatment group will haveapproximately the same number of individuals injected from each of thecollection plants.

After injection, aphids will be released into a petri dish onto a favabean leaves that have stems in an Eppendorf tube filled with 1 ml water.Aphid survival will be monitored daily, and dead aphids will be removedwhen they are found. The number of offspring from each group will becounted and fecundity will be measured as the number of offspring (F1's)produced per aphid at each time point.

In select experiments, development will be measured in groups ofoffspring from each treatment group by noting the developmental stage ofoffspring each day (1^(st), 2^(nd), 3^(rd), 4^(th), and 5^(th) instar).Development will be also measured by imaging aphids at 4 dayspost-collection and determining their area. The template forsynthesizing dsRNAs will be the cDNA reverse-transcribed from the mRNA.RNA will be extracted from one 5th instar A. pisum (LSR-1 strain) and isquantified by Nanodrop (Thermo fisher scientific). ˜100 μg of total RNAwill be added as template into the reverse-transcription reaction usingSuperscript IV kit (Thermo Fisher Scientific) following manufacturer'sprotocol.

To amplify the templates for the dsRNAs, the cDNA will be diluted100-fold and used in the following PCRs. The PCR reactions (25 μl finalvolume) contain 12.5 μL of Go Taq Green 2X mix (Promega), 0.2 μl offorward primer (Table 12), 0.2 μl of reverse primer (Table 12), and 12.1μl of 100-fold diluted cDNA. PCR reactions are performed using followingconditions: 1) 95° C. for 2 minutes, 2) 95° C. for 20 seconds, 3) 55° C.for 15 seconds, 4) 72° C. for 30 seconds, 5) repeat steps 2-4 35×, 6)72° C. for 5 minutes. The sizes of PCR amplified products are verifiedby electrophoresis on 1.5% agarose and the expected-size bands are cutand purified by QIAquick DNA purification kit (Qiagen). The dsRNAs willbe synthesized in vitro using T7 MEGAscript kit (Ambion, Thermo FisherScientific; Cat# AM1334) following manufacturer's protocol. Theconcentration of dsRNA is measured by Nanodrop (Thermo FisherScientific).

TABLE 12 Cact dsRNA sequences: Target product gene Forward primerReverse primer size Mpcactus taatacgactcactat taatacgactcactata 487agggTACACCCATTGT gggCCACTGTCCAAG GTGCACCT GCAATTTT (SEQ ID NO: 99) (SEQID NO: 100)

The status of Buchnera in aphid samples will be assessed by PCR. Aphidsadults from the negative control and phage treated will be firstsurface-sterilized with 70% ethanol for 1 min, 10% bleach for 1 min andthree washes of ultrapure water for 1 min. Total DNA will be extractedfrom each individual (whole body) using an Insect DNA Kit (OMEGA,Bio-tek) according to the manufacturer's protocol. The primers forBuchnera, forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 97) andreverse primer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 98), are designedbased on 23S-5S rRNA sequences obtained from the Buchnera genome(Accession Number: GCA_000009605.1) (Shigenobu, et al., Nature 200.407,81-86) using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). The PCRamplification cycles include an initial denaturation step at 95° C. for5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 60s, and a final extension step of 10 min at 72° C. Amplification productsfrom rifampicin treated and control samples will be analyzed on 1%agarose gels, stained with SYBR safe, and visualized using an imagingSystem. dsRNA treated aphids are expected to show a reduction ofBuchnera specific genes.

The expression of Cact and survival rates of aphids treated with Cactusare compared to the aphids treated with the negative control. Theexpression of Cact and survival rate of aphids treated with dsRNA toCact are expected to decrease as compared to the control treated aphids.

Example 17: Aphids Treated with a Fungi Solution that Stimulated anInsect Immune Response to Decrease Fitness

This Example demonstrates the treatment of aphids with a solution ofPichia pastoris, a yeast strain that resulted in decreased aphidfitness. Although many of the innate immune system pathways present ininsects are absent in aphids, the key players that recognize and induceimmune responses to fungi remain intact. Specifically, the presence offungi induces the cleavage of the aphid serine protease, Persephone,which then activates the Toll pathway. Next, Cactus is phosphorylatedand Dorsal is released and translocated to the nucleus which activatesthe expression and secretion of antimicrobial peptides, lysozymes, andprophenoloxidase that ultimately affect bacterial populations endogenousto the aphid. This Example demonstrates that the effect of the solutionof Pichia pastoris on aphids was mediated through the modulation ofbacterial populations endogenous to the aphid that were sensitive to theincrease in the Aphid immune response generated by the presence of theyeast. One targeted bacterial strain was Buchnera.

Aphids are agricultural insect pests causing direct feeding damage tothe plant and serving as vectors of plant viruses. In addition, aphidhoneydew promotes the growth of sooty mold and attracts nuisance ants.The use of chemical treatments, unfortunately still widespread, leads tothe selection of resistant individuals whose eradication becomesincreasingly difficult.

Therapeutic Design

P. pastoris was delivered using two different methods: Fava bean leafperfusion and air brush spraying onto fava bean leaves. For eachexperiment, there were two experimental groups: 1) treatment with wateras a negative control; and 2) treatment with P. pastoris in water.Treatment methods and doses are described herein in the ExperimentalDesign section.

Leaf Perfusion Experimental Design

Aphids (LSR-1 strain, Acyrthosiphon pisum) were grown on fava beanplants (Vroma vicia faba from Johnny's Selected Seeds) in aclimate-controlled incubator (16 h light/8 h dark photoperiod; 60±5% RH;25±2° C.). Prior to being used for aphid rearing, fava bean plants weregrown in potting soil at 24° C. with 16 h of light and 8 h of darkness.To limit maternal effects or health differences between plants, 5-10adults from different plants were distributed among 10 two-week-oldplants, and allowed to multiply to high density for 5-7 days. Forexperiments, first and second instar aphids were collected from healthyplants and divided into two different treatment groups: 1) those allowedto feed on leaves perfused with water; and 2) those allowed to feed onleaves perfused with a P. pastoris solution in water.

The “protease wild-type” ade2 knockout strain (Strain 1) from the PichiaPink System (Thermo Fisher Scientific) was used for experiments. P.pastoris was grown in YPD overnight at 30° C., and the following day,the culture was washed once with water and resuspended in water to afinal OD₆₀₀ of 0.918 for the first leaf perfusion on day 0 or OD₆₀₀ of5.58 for the second leaf perfusion on day 3 of the experiment. For eachleaf perfusion, approximately 1 ml of the P. pastoris solution or water(negative control) was injected into a fava bean leaf. The stem of theleaf was then placed in a 1.5 ml Eppendorf that was sealed withparafilm. The leaf stem was then placed into a deep petri dish (FisherScientific, Cat# FB0875711) and aphids were applied to the leaves of theplant and allowed to feed. Leaves were changed on day 3 of theexperiment and old leaves were replaced with new leaves perfused with P.pastoris (at the indicated OD₆₀₀) or water.

For each treatment, 62-63 aphids were placed onto each leaf. Aphids weremonitored daily for survival and dead aphids were removed when they werediscovered. In addition, the developmental stage (1^(st), 2^(nd),3^(rd), 4^(th), and 5^(th) instar) was determined daily throughout theexperiment.

After 6 days of treatment, DNA was extracted from multiple aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 101) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 102) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 103) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 104) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Treatment with a Yeast Solution Delayed Progression of Aphid Development

LSR-1 first and second instar aphids were divided into two treatmentgroups as defined in Leaf Perfusion Experimental Design. Aphids weremonitored daily and the number of aphids at each developmental stage wasdetermined. By day 6 of the experiment, approximately 30% of aphidsfeeding on leaves perfused with water reached the fifth instar stage(FIG. 1). In contrast, only approximately 3% of aphids feeding on leavesperfused with P. pastoris reached the fifth instar stage by day 6 of theexperiment (FIG. 1). The majority of aphids feeding on the leavesperfused with P. pastoris that survived to day 6 were at the 4th instarstage (˜5%) (FIG. 1). These data indicated that P. pastoris treatmentslowed aphid development.

Treatment with a Yeast Solution Increased Aphid Mortality

Survival of aphids was also measured during the treatments.Approximately 55.5% of aphids feeding on leaves perfused with watersurvived to day 6 of the experiment (FIG. 2). In contrast, aphidsfeeding on leaves perfused with P. pastoris rapidly began to die at day2 post-treatment and by day 6, only 11% of aphids were alive (FIG. 2).These data indicated that P. pastoris treatment delivered through leafperfusion resulted in a significant (p<0.0001) increase in aphidmortality.

To test whether P. pastoris treatment specifically resulted in loss ofBuchnera in aphids, and that this loss impacted aphid fitness, DNA wasextracted from aphids in each treatment group after 6 days of treatmentand qPCR was performed to determine the Buchnera/aphid copy numbers.Results were inconclusive due to the low number of aphids remaining inthe P. pastoris treatment group at day 6 and the lack of extractableBuchnera DNA.

Airbrush Spraying Experimental Design:

Aphids (LSR-1 strain, Acyrthosiphon pisum) were grown on fava beanplants as described herein. For experiments, first and second instaraphids were collected from healthy plants and divided into 2 differenttreatment groups: 1) aphids placed on leaves sprayed with water(negative control), and 2) aphids placed on leaves sprayed with P.pastoris.

The “protease wild-type” ade2 knockout strain (Strain 1) from the PichiaPink System (Thermo Fisher Scientific) was used for experiments. P.pastoris was grown in YPD overnight at 30° C. and the following day, theculture was washed once with water and resuspended in water to a finalOD600 of 0.918 for the first and second leaf spraying (on day 0 and 3)and OD600 of 5.58 for the final leaf spraying on day 6 of theexperiment. For the treatments, fava bean leaves were cut and the stemswere placed into a 1.5 ml Eppendorf tube with sterile water and sprayedon both sides using an airbrush with water or P. pastoris at theconcentration indicated above. The leaves were then placed in a deeppetri dish (Fisher Scientific, Cat# FB0875711) and aphids were appliedto the leaves of the plant and allowed to feed. On day 3 and 6 of theexperiment, old leaves were replaced with new, freshly sprayed leaves.

Each treatment group received approximately the same number ofindividuals from each of the collection plant. For each treatment, 30aphids were placed onto each leaf. Each treatment was performed induplicate for a total of 60 aphids/treatment group. Aphids weremonitored daily for survival, and dead aphids were removed when theywere discovered. In addition, the developmental stage (1st, 2nd, 3rd,4th, and 5th instar) was determined daily throughout the experiment.

After 6 and 9 days of treatment, DNA was extracted from multiple aphidsfrom each treatment group and qPCR for quantifying Buchnera levels wasdone as described herein.

Treatment with a Yeast Solution Did not Greatly Impact Aphid Development

LSR-1 first and second instar aphids were divided into two treatmentgroups as defined in the Airbrush Spraying Experimental Design describedherein. Aphids were monitored daily and the number of aphids at eachdevelopmental stage was determined. By 6 days post-treatment, aphidsfrom both treatment groups began reaching the 5th instar stage (FIG. 3).By day 9 post-treatment, nearly all remaining aphids in each groupreached maturity (FIG. 3). These data indicated that there was littledifference in development between aphids treated with water or P.pastoris.

A Yeast Solution Treatment Via Spraying Resulted in Increased AphidMortality

Survival of aphids was also measured during the leaf sprayingexperiments. Throughout the course of the experiment, only a few aphidsfeeding on leaves sprayed with water died, whereas a greater number ofaphids died at each time point in the P. pastoris treatment group (FIG.4). Specifically, on day 2 post-treatment 91% of water-treated aphidsremained alive, while only 80% of P. pastoris-treated aphids remainedalive. This trend continued through days 3, 6, and 7 of the experimentwhere 82%, 66%, and 60% of water-treated aphids were alive at each timepoint, respectively, in contrast to only 73%, 55%, and 49% of P.pastoris-treated aphids were alive at each time point, respectively(FIG. 4). These data indicated that P. pastoris treatment delivered byairbrush spraying resulted in higher mortality compared to treatmentwith water alone.

A Yeast Solution Treatment Reduced Buchnera Titers in Aphids

To test whether P. pastoris delivered through airbrush spraying,specifically resulted in loss of Buchnera in aphids, and that this lossimpacted aphid fitness, DNA was extracted from aphids in each treatmentgroup after 6 and 9 days of treatment and qPCR was performed todetermine the Buchnera/aphid copy numbers. At 6 days post-treatment themean Buchnera/aphid ratios were 47.5 in aphids feeding on leaves sprayedwith water (FIG. 5). In contrast, mean Buchnera/aphid ratios wereapproximately 1.2-fold lower (˜39) in aphids feeding on leaves sprayedwith P. pastoris (FIG. 5). At 9 days post-treatment however, the ratiosof Buchnera/aphid DNA copies were similar in both treatment groups (FIG.5).

These data indicated that spraying P. pastoris onto fava bean leavesdecreased endosymbiotic Buchnera in aphids after 6 days post-treatment.It was possible that P. pastoris-treated aphids that survived to 9 dayspost-treatment were able to overcome the detrimental effects of theincreased immune response and could explain why these aphids had similarBuchnera titers to the water treated controls. Nonetheless, examinationof the median Buchnera/aphid copies on day 9, revealed a trend fordecreased Buchnera in the P. pastoris treatment group.

Confirmation of that P. pastoris Upregulates the Immune Response inAphids

Given that treatment with P. pastoris resulted in decreased aphidfitness and lower titers of the aphid endosymbiont, Buchnera, the nextexperiment is to confirm that P. pastoris is upregulating the immuneresponse. To test this, RNA has been isolated from aphids in each of theexperiments described herein (Leaf Perfusion Experimental Design andAirbrush Spraying Experimental Design) and the expression of genesinvolved in the fungal immune response pathway will be measured(Persephone, Cactus, and Dorsal) as described in Gerardo et al., 2010,Genome Biology, 11:R21, https://doi.org/10.1186/gb-2010-11-2-r21. It isexpected to see upregulation of the genes involved in this pathway.

Together, the data described in these Examples demonstrated the abilityto kill and decrease the development, longevity, and endogenousbacterial populations, e.g., fitness, of aphids by treating them with P.pastoris which likely resulted in activation of the aphid immuneresponse.

Example 18: Aphids Treated with a Solution of a Peptide Nucleic Acid

This Example demonstrates that the treatment of aphids with a peptidenucleic acid (PNA) to BCR-4 fused to a cell penetrating peptide (CPP)(Cermenati et al., 2011; Zhou et al., 2015), herein referred to as PNAto BCR-4 or BCR-4 PNA, resulted in knock-down of BCR-4 gene expressionand reduction of aphid fitness.

BCR-4 is one of several cysteine-rich secreted peptides expressed inbacteriocytes of the pea aphid, Acyrthosiphon pisum. The obligate aphidbacterial symbiont, Buchnera, is housed inside of the bacteriocytes.BCR-4 has sequence homology to many of the nodule cysteine rich (NCRs)peptides involved in keeping plant bacterial symbiont numbers in check(Pan and Wang, 2017 Nature Plants and Durgo et al., 2015 Proteomics).Given the sequence similarity of BCR-4 and the NCRs, this Exampledemonstrates that BCR-4 played a similar role in maintainingendosymbiont homeostasis inside the bacteriocyte by disrupting BCR-4though gene knockdown to dysregulate Buchnera, thereby negativelyaffecting aphid fitness.

Therapeutic Design

The BCR-4 PNA was delivered either by microinjection or through favabean leaf perfusion. For microinjection experiments, injection solutionswere either water (negative control) or BCR-4 PNA in water. For leafperfusion studies, fava bean leaves were perfused with water (negativecontrol) or with BCR-4 PNA in water. Each experimental delivery designis explained in detail below.

Microinjection Delivery Experimental Design

Microinjection was performed using NanoJet III Auto-Nanoliter Injectorwith an in-house pulled borosilicate needle (Drummond Scientific;Cat#3-000-203-G/XL). Aphids were transferred using a paint brush to atubing system connected to vacuum which held them in place during theinjection. The injection site was at the ventral thorax of the aphid.The injection solutions were water (negative control) or 321 ng/μl ofBCR-4 PNA in water. The injection volume was 20 nl for adult (4th and5th instar) aphids at a rate of 2 nl/sec. Each treatment group hadapproximately the same number of individuals injected from each of thecollection plants. After injection, aphids were released into a petridish onto fava bean leaves that had stems in an Eppendorf tube filledwith 1 ml water. Survival and fecundity were monitored over the courseof the experiment.

Aphid Rearing and Maintenance:

Aphids LSR-1 (which harbor only Buchnera), A. pisum were grown on favabean plants (Vroma vicia faba from Johnny's Selected Seeds) in aclimate-controlled incubator (16 h light/8 h dark photoperiod; 60±5% RH;25±2° C.). Prior to being used for aphid rearing, fava bean plants weregrown in potting soil at 24° C. with 16 h of light and 8 h of darkness.To limit maternal effects or health differences between plants, 5-10adults from different plants were distributed among 10 two-week-oldplants, and allowed to multiply to high density for 5-7 days. Forexperiments, fourth and fifth instar aphids were collected from healthyplants and divided into two treatment groups: 1) water (negativecontrol) or 2) BCR-4 PNA in water.

BCR-4 PNA Synthesis

BCR-4-CPP was synthesized by PNA bio and the sequence isYGRKKRRQRRR-CGTACAATAATCTCATGG; SEQ ID NOs: 105 and 106. The sequence ofthe CPP (Tat) is

YGRKKRRQRRR; SEQ ID NO: 106. The PNA was dissolved in 80% Acetonitrileand 20% Water supplemented with 0.1% Trifluoroacetic acid (TFA). Oncedissolved, the PNA was aliquoted [32.1 μg/5 nmol per aliquot], airdried, and stored at −20° C. Working solutions of the PNA were made inwater at 50 uM.

After 7 days of treatment, RNA was extracted from the remaining aphidsin each treatment group. Briefly, the aphid body surface was sterilizedby dipping the aphid into a 6% bleach solution for approximately 5seconds. Aphids were then rinsed in sterile water and total RNA wasextracted from each individual aphid using the RNA extraction kit(Qiagen miRNeasy kit) according to manufacturer's instructions. RNAconcentrations were measured using a nanodrop nucleic acidquantification. BCR-4 relative expression was measured by RT-qPCR. Theprimers used were ApBCR-4F (CTCTGTCAACCACCATGAGATTA; SEQ ID NO: 107) andApBCR-4R (TGCAGACTACAGCACAATACTT; SEQ ID NO: 108). The internalreference gene primers were for Actin (housekeeping gene). The forwardsequence was GATCAGCAGCCACACACAAG; SEQ ID NO: 109 and the reversesequence was TTTGAACCGGTTTACGACGA; SEQ ID NO: 110. RT-qPCR was performedusing a qPCR amplification ramp of 1.6 degrees C./s and the followingconditions: 1) 48° C. for 30 min, 2) 95° C. for 10 minutes, 3) 95° C.for 15 seconds, 4) 60° C. for 1 minute, 5) repeat steps 3-4 40×, 6) 95°C. for 15 seconds, 7) 60° C. for 1 minute, 8) ramp change to 0.15degrees C./s, 9) 95° C. for 1 second. RT-qPCR data was analyzed usinganalytic (Thermo Fisher Scientific, QuantStudio Design and Analysis)software.

Microinjection with a PNA Against the BCR-4 Gene Resulted in DecreasedExpression of BCR-4

To test whether a PNA against BCR-4 delivered by microinjection resultsin decreased BCR-4 gene expression in aphids, aphids were injected withwater (control) or BCR-4 PNA. After 7 days of treatment, RNA wasextracted from aphids in each treatment group and RT-qPCR was performed.Aphids microinjected with water had approximately 2-fold higherexpression of BCR-4 compared to aphids injected with BCR-4 PNA (FIG. 6),indicating that injection of BCR-4 PNA resulted in knockdown of BCR-4.

Treatment with a PNA to BCR-4 Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments. At mosttime points during the experiment, there were more control (waterinjected) aphids alive compared to aphids injected with the PNA to BCR-4(FIG. 7). These data indicated that there was a slight decrease insurvival upon injection with a PNA to BCR-4.

Treatment with a PNA to BCR-4 Reduced Aphid Fecundity

The number of offspring produced from aphids in each treatment group wasalso assessed during the experiment and fecundity was represented as thenumber of offspring produced per adult at each time point assessed.Overall, there was a trend for control (water-injected) aphids producingmore offspring compared to those injected with the PNA to BCR-4.Specifically, at 3 and 7 days post-treatment, aphids in the watertreated group produced an average of 5 offspring/adult whereas aphids inthe BCR-4 PNA treatment group only produced 4 offspring/adult (FIG. 8).These data indicated that treatment with BCR-4 PNA resulted in a slightdecrease in aphid fecundity.

Leaf Perfusion Experimental Design

Aphids (LSR-1 strain, Acyrthosiphon pisum) were grown on fava beanplants (Vroma vicia faba from Johnny's Selected Seeds) in aclimate-controlled incubator (16 h light/8 h dark photoperiod; 60±5% RH;25±2° C.). Prior to being used for aphid rearing, fava bean plants weregrown in potting soil at 24° C. with 16 h of light and 8 h of darkness.To limit maternal effects or health differences between plants, 5-10adults from different plants were distributed among 10 two-week-oldplants and allowed to multiply to high density for 5-7 days. Forexperiments, first and second instar aphids were collected from healthyplants and divided into two different treatment groups: 1) those allowedto feed on leaves perfused with water, and, 2) those allowed to feed onleaves perfused with a BCR-4 PNA solution in water.

The BCR-4 PNA fused to a CPP was synthesized as described herein (seeMicroinjection Delivery Experimental Design BCR-4 PNA synthesis).

For each leaf perfusion, approximately 1 ml of water (negative control)or a 1 uM BCR-4 PNA solution was injected into a fava bean leaf. Thestem of the leaf was then placed in a 1.5 ml Eppendorf that was sealedwith parafilm. The leaf stem was then placed into a deep petri dish(Fisher Scientific, Cat# FB0875711) and aphids were applied to theleaves of the plant and allowed to feed. Old leaves were replaced withnew, freshly injected leaves every 2-3 days throughout the experiment.For each treatment, 60 aphids were placed onto each leaf. Aphids weremonitored daily for survival and dead aphids were removed when they werediscovered. In addition, the developmental stage (1st, 2nd, 3rd, 4th,and 5th instar) was determined daily throughout the experiment.

After 5 and 6 days of treatment, DNA was extracted from dead aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 101) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 102) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 103) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 104) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

At 7 post-treatment, RNA was extracted from live aphids and RT-pPCR wasdone as described above (see Microinjection Delivery ExperimentalDesign) to quantify expression of BCR-4.

Leaf Perfusion Treatment with BCR-4 PNA Delayed Progression of AphidDevelopment

LSR-1 first and second instar aphids were divided into two groups asdefined in Leaf Perfusion Experimental Design. Aphids were monitoreddaily and the number of aphids at each developmental stage wasdetermined. At several time points during the experiment, developmentwas delayed in aphids treated with the BCR-4 PNA. For example, on day 2post-treatment, approximately 19% of aphids feeding on leaves perfusedwith water were in the 3rd instar stage (FIG. 9). In contrast, only 8%of aphids feeding on leaves perfused with the PNA to BCR-4 were in the3rd instar stage. These data indicated that treatment with a PNA toBCR-4 slowed aphid development.

Leaf Perfusion Treatment with BCR-4 PNA Increased Aphid Mortality

Survival was also monitored throughout the course of treatment. By 7days post-treatment, 53% of aphids in the water (control) treatmentgroup remained alive (FIG. 10). In contrast, only 30% of aphids in thePNA BCR-4 treatment group were alive on day 7 (FIG. 10). These datashowed that treatment with a PNA to BCR-4 resulted in increased aphidmortality.

Leaf Perfusion Treatment with BCR-4 PNA Increased Buchnera Titers inAphids

To test whether BCR-4 PNA delivered through leaf perfusion, specificallyresulted affecting Buchnera in aphids, and that this impacted aphidfitness, DNA was extracted from dead aphids in each treatment groupafter 5 and 6 days post-treatment, and qPCR was performed to determineBuchnera tiers in each treatment group. While aphids feeding on leavesperfused with water had a mean ratio of approximately 20 Buchnera/aphidcopies, aphids feeding on leaves perfused with the PNA to BCR-4 hadsubstantially higher Buchnera/aphid copies (approximately 57), see FIG.11. These data indicate that treatment with a PNA to BCR-4 leads to amisbalance in Buchnera titers.

Treatment with a PNA Against BCR-4 Via Leaf Perfusion Knocked Down BCR-4

To assess whether BCR-4 expression was reduced in aphids feeding onleaves perfused with a PNA to BCR-4, RNA was isolated from living aphidsafter 7 days of treatment and RT-qPCR was performed. The mediantranscript levels of BCR-4 in aphids treated with a PNA to BCR-4 wereapproximately 3-fold lower compared to aphids treated with water alone(FIG. 12), confirming that the PNA to BCR-4 knocked down BCR-4expression.

Together, these data demonstrated the ability to kill and decrease thedevelopment, fecundity, and longevity, (e.g., fitness), of aphids bytreating them with a PNA targeting a gene expressed in bacteriocytes(BCR-4) to control the population of endosymbiont Buchnera.

Example 19: Aphids Treated with a Solution of dsRNA Against BacteriocyteTransporters

This Example demonstrates that the treatment of aphids withdouble-stranded RNA (dsRNA) resulted in the knock-down of some essentialgenes, including glutamine transporter 1 gene (ApGLNT1), which has beenidentified in the bacteriocytes in the aphid, Acyrthosiphon pisum. Theglutamine transporter is responsible for glutamine uptake from the aphidhemolymph into the bacteriocytes where the obligate endosymbiont,Buchnera aphidicola, is located. The combined biosynthetic capability ofthe holobiont (A. pisum and Buchnera) is sufficient for biosynthesis ofall twenty protein coding amino acids, including amino acids that aphidsalone cannot synthesize. Blocking the glutamine uptake by deactivatingor silencing (e.g. RNAi by dsRNA) the glutamine transporter negativelyaffected amino acid and protein synthesis in the bacteriocytes and inthe entire aphid, thereby negatively affecting their fitness.

Therapeutic Design

5^(th) instar LSR-1 aphids were microinjected. The injection solutionswere either dd-water (negative control) or dsRNA diluted in dd-water atvarious concentrations (8 or 60 ng/aphid; see below).

Aphid Rearing and Maintenance

Aphids LSR-1 (which harbor only Buchnera), A. pisum were grown on favabean plants (Vroma vicia faba from Johnny's Selected Seeds) in aclimate-controlled incubator (16 h light/8 h dark photoperiod; 60±5% RH;25±2° C.). Prior to being used for aphid rearing, fava bean plants weregrown in potting soil at 24° C. with 16 h of light and 8 h of darkness.To limit maternal effects or health differences between plants, 5-10adults from different plants were distributed among 10 two-week-oldplants, and allowed to multiply to high density for 5-7 days. Forexperiments, fifth instar aphids were collected from healthy plants anddivided into 2 different treatment groups: 1) water (negative control)or 2) dsRNA against ApGLNT1 (at the concentrations indicated herein).

Microinjection Delivery Experimental Design

Microinjection was performed using NanoJet III Auto-Nanoliter Injectorwith an in-house pulled borosilicate needle (Drummond Scientific;Cat#3-000-203-G/XL). Aphids were transferred using a paint brush to atubing system connected to vacuum which held the aphid in place duringthe microinjection. The injection site was at the ventral thorax of theaphid. The injection volume was 20 nl for adult aphids at a rate of 2nl/sec. Each treatment group had approximately the same number ofindividuals injected from each of the collection plants.

After injection, aphids were released into a petri dish onto a fava beanleaves that had stems in an Eppendorf tube filled with 1 ml water. Aphidsurvival was monitored daily, and dead aphids were removed when theywere found. The number of offspring from each group was counted andfecundity was measured as the number of offspring (F1's) produced peraphid at each time point.

In select experiments, development was measured in groups of offspringfrom each treatment group by noting the developmental stage of offspringeach day (1^(st), 2^(nd), 3^(rd), 4^(th), and 5^(th) instar).Development was also measured by imaging aphids at 4 dayspost-collection and determining their area. The template forsynthesizing dsRNAs was the cDNA reverse-transcribed from the mRNA. RNAwas extracted from one 5th instar A. pisum (LSR-1 strain) and wasquantified by Nanodrop (Thermo fisher scientific). ˜100 μg of total RNAwas added as template into the reverse-transcription reaction usingSuperscript IV kit (Thermo Fisher Scientific) following manufacturer'sprotocol.

To amplify the templates for the dsRNAs, the cDNA was diluted 100-foldand used in the following PCRs. The PCR reactions (25 μl final volume)contain 12.5 μL of Go Taq Green 2X mix (Promega), 0.2 μl of forwardprimer (Table 12), 0.2 μl of reverse primer (Table 12), and 12.1 μl of100-fold diluted cDNA. PCR reactions were performed using followingconditions: 1) 95° C. for 2 minutes, 2) 95° C. for 20 seconds, 3) 55° C.for 15 seconds, 4) 72° C. for 30 seconds, 5) repeat steps 2-4 35×, 6)72° C. for 5 minutes. The sizes of PCR amplified products were verifiedby electrophoresis on 1.5% agarose and the expected-size bands were cutand purified by QIAquick DNA purification kit (Qiagen). The dsRNAs weresynthesized in vitro using T7 MEGAscript kit (Ambion, Thermo FisherScientific; Cat# AM1334) following manufacturer's protocol. Theconcentration of dsRNA was measured by Nanodrop (Thermo FisherScientific).

TABLE 12 Accession numbers and primers for dsRNA syntheses. Gene gene IDForward primer Reverse primer ApGLNT1 ACYPI001018 TAATACGACTCACTTAATACGACTCAC ATAGGGCAATTACA TATAGGGCCGCTCT AAAGGACGGCAG AGGAACACCGTAT(SEQ ID NO: 111) (SEQ ID NO: 112)

At the indicated time point post-treatment, DNA and/or RNA was extractedfrom aphids in each treatment group. Briefly, the aphid body surface wassterilized by dipping the aphid into a 6% bleach solution forapproximately 5 seconds. Aphids were then rinsed in sterile water andtotal DNA or RNA was extracted from each individual aphid using eitherthe DNA or RNA extraction kit (Qiagen, DNeasy or miRNeasy kit,respectively) according to manufacturer's instructions. DNA and RNAconcentrations were measured using a nanodrop nucleic acidquantification. Buchnera and aphid DNA copy numbers were measured byqPCR. The primers used for Buchnera were Buch_groES_18F(CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 101) and Buch_groES_98R(CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 102) (Chong and Moran, 2016 PNAS).The primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQID NO: 103) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 104)(Chong and Moran, 2016 PNAS). qPCR was performed using a qPCRamplification ramp of 1.6 degrees C./s and the following conditions: 1)95° C. for 10 minutes, 2) 95° C. for 15 seconds, 3) 55° C. for 30seconds, 4) repeat steps 2-3 40×, 5) 95° C. for 15 seconds, 6) 55° C.for 1 minute, 7) ramp change to 0.15 degrees C./s, 8) 95° C. for 1second. qPCR data was analyzed using analytic (Thermo Fisher Scientific,QuantStudio Design and Analysis) software. ApGLNT1 relative expressionwas measured by RT-qPCR. The primers used for ApGLNT1 wereACYPI001018-fwd (CCTGAAATCGACGGGGTCC; SEQ ID NO: 113) andACYPI001018-rev (AGATCGGCAACATCTGTTCGT; SEQ ID NO: 114) (both designedby NCBI pick primers). The internal reference gene was Actin(housekeeping gene). The primers used for Actin were Actin-F(GATCAGCAGCCACACACAAG; SEQ ID NO: 109) and Actin-R(TTTGAACCGGTTTACGACGA; SEQ ID NO: 110) RT-qPCR was performed using aqPCR amplification ramp of 1.6 degrees C./s and the followingconditions: 1) 48° C. for 30 min, 2) 95° C. for 10 minutes, 3) 95° C.for 15 seconds, 4) 60° C. for 1 minute, 5) repeat steps 3-4 40×, 6) 95°C. for 15 seconds, 7) 60° C. for 1 minute, 8) ramp change to 0.15degrees C./s, 9) 95° C. for 1 second. RT-qPCR data was analyzed usinganalytic (Thermo Fisher Scientific, QuantStudio Design and Analysis)software.

Microinjection with dsRNA Knocked-Down the Bacteriocyte Transporter GeneExpression in Aphids

The preliminary experiment assessed whether injecting dsRNA into aphidswould result in decreased gene expression. Adult aphids were injectedwith 8 ng dsRNA or water (as a negative control). On two dayspost-injection, RNA was extracted from the remaining aphids in eachtreatment group and RT-qPCR was performed to quantify expression ofApGLNT1. Aphids microinjected with the negative control solution (water)had high relative expression of the ApGLNT1 gene. In contrast, aphidadults microinjected with the dsRNA of ApGLNT1 had a drastic, andsignificant, reduction of ApGLNT1 gene expression (FIG. 13), indicatingthat dsRNA microinjection treatment decreased expression of ApGLNT1gene.

Microinjection of dsRNA-ApGLNT1 Resulted in Increased Aphid Mortality

To assess the effect of dsRNA-ApGLNT1 microinjection on insect fitness,LSR-1 fifth instar aphids were injected with 60 ng dsRNA and survivalwas monitored for 5 days. At 3 days post-injection, approximately 72% ofwater-injected aphids were alive (FIG. 14). In contrast, only 52% ofdsRNA-injected aphids were alive (FIG. 14). At days 4 and 5post-injection, there were significantly more aphids alive in thewater-injected group compared to the dsRNA-injected group. Approximately62% and 51% of water-injected aphids were alive on days 4 and 5,respectively, whereas only 30% and 12.5% of dsRNA-injected aphids werealive on day 4 and 5, respectively (FIG. 14). These data indicated thatdsRNA against ApGLNT1 significantly (p=0.0004) increased aphid mortalitycompared to water-injected controls.

Microinjection of dsRNA-ApGLNT1 Resulted in Decreased Buchnera Titers

To test whether the decrease in survival and fitness was due todecreased number of endosymbionts, DNA was extracted from living aphidsat 5 days post-injection and qPCR was performed to quantify the amountof Buchnera present in the aphid. Aphids microinjected with water hadmean ratios of approximately 11 Buchnera/aphid copies (FIG. 15). Incontrast, Buchnera/aphid copies was approximately 1.28 times lower inaphids injected with the dsRNA against the ApGLNT1 (FIG. 15). These datashowed that microinjection of dsRNA-ApGLNT1 resulted in decreasedBuchnera titers which led to decreased aphid fitness.

Development was Delayed in Offspring of Aphids Microinjected withdsRNA-ApGLNT1

On day 3 post-injection, 40 offspring (first instars) from eachtreatment group were transferred to their own artificial feeding system(a petri dish containing a fava bean leaf stem put into a 1.5 mlEppendorf tube sealed with parafilm) and development was monitored overtime. Overall, development was delayed in offspring taken from adultsinjected with dsRNA-ApGLNT1. By day 4 post offspring transfer,approximately 22.5% of offspring from water-injected aphids beganreaching the 5th instar stage (FIG. 4A). In contrast, on day 4, only7.5% of offspring from adults injected with dsRNA-ApGLNT1 reached the5th instar stage (FIG. 16A). Additionally, aphid areas were measured onday 4 by imaging each aphid in each group. While the average size ofoffspring from water-injected adults was 0.55 mm², offspringdsRNA-ApGLNT1-injected adults were significantly smaller (p=0.009) andaveraged 0.4 mm² (FIG. 16B). These data indicated that treatment ofadults with dsRNA-ApGLNT1 resulted in offspring with severely delayeddevelopment.

Example 20: Production of Transgenic Plants Expressing dsRNAs thatTarget Multiple Pathways to Destabilize Insect-Symbiont HomeostasisThrough Treatment of Aphids with the Transgenic Plants

This Example demonstrates the ability to genetically modify Nicotianatabacum to produce dsRNA for delivery to aphids to affectinsect-symbiont homeostasis. Genetic constructs that stably expressdsRNA in Nicotiana tabacum are delivered to the plant using transgenicAgrobacterium tumefaciens that will carry the plasmid to the plant.

Experimental Design

Several genes will be targeted for knockdown in multiple pathways thatare critical for the symbiotic relationship between the aphids and theirobligate endosymbiotic bacteria, Buchnera. Specifically, the glutaminetransporter of the bacteriocytes (GlnT1), ultrabithorax (Ubx), betaalanine synthase (bAS), and cactus (Cact) are targeted. GlnT1 is aglutamine transporter that is used for the import of glutamine into thebacteriocytes, and the downstream products of glutamine are essentialfor the synthesis of essential amino acids by Buchnera. Ubx is a geneinvolved in both the general development of the aphids, as well as theformation of the bacteriocytes which will house Buchnera. bAS is anaphid gene required to synthesize beta Alanine which is a precursor forthe synthesis of vitamin B5 by Buchnera. Cact is the negative regulatorof the Toll pathway, which is the primary immunity pathway in aphids.Lowering the levels of Cact could upregulate the systemic immuneresponses leading to the dysregulation of the Buchnera levels.

Generating a Plasmid Containing dsRNA Expression Cassette:

A shuttle vector between E. coli and A. tumefaciens will be used tocarry the dsRNA expression cassette, which includes the cauliflowermosaic virus 35S promoter (pCaMV 35S) upstream of the dsRNA expressingsequence. The dsRNA expressing sequence includes the sense sequencefollowed by the antisense sequence of a region of the target aphid geneconnected by a small hairpin loop sequence (FIG. 17).

The dsRNA expression cassette will then be placed in a shuttle vectorfor E. coli and A. tumefaciens (FIG. 18). The plasmid will also containboth Kanamycin and Gentamycin resistance cassettes which can be used asselection markers. A transcription terminator will be placed after thedsRNA expression transcript to eliminate runaway transcription.

The dsRNA expressing sequences for various genes (GlnT1, Ubx, bAS, andCact) can be introduced into the vector via Gibson assembly. First, thesense and the antisense amplicons with overhangs that match theneighboring regions in the plasmid will be generated using theappropriate primers in a PCR. Specifically, the left overhang of thesense strand will have region of homology (˜30 bp) to the pCaMV 35S, andthe right overhang of the antisense strand will have region of homology(˜30 bp) to the 35S terminator region. The right overhang of the sensestrand and the left overhang of the antisense strands will haveoverlapping pieces of each other with the inclusion of a small hairpinregion (acacgt, SEQ ID NO: 115). The primer sequences to produce theamplicons are shown in Table 13.

TABLE 13 List of primers to generate amplicons for the Gibson assemblyto generate hairpin dsRNA sequences against A. pisum genes. Each targetgene's region will be amplified as either sense or antisense withflanking regions that are homologous to the plasmid backbone for Gibsonassembly. Primer name Primer sequence ApGlnT1 sensectacaaatctatctctcctaggCAATTACAAAAGGACGGCAG (SEQ ID NO: 116) fwd ApGlnT1sense ttacaaactgggaagaacctggagacgtgCCGCTCTAGGAACACCGTAT (SEQ ID rev NO:117) ApGlnT1 ttacaaactgggaagaacctggaacacgtcCCGCTCTAGGAACACCGTAT (SEQantisense fwd ID NO: 118) ApGlnT1agaaactagagcttgtcgatcgttaattaaCAATTACAAAAGGACGGCAG (SEQ ID antisense revNO: 119) ApUbx sense fwd ctacaaatctatctctcctaggTTTTACCGTCACAGGCATCA (SEQID NO: 120) ApUbx sense revacgagtgctgaagtccctagccagacgtgtTCGTGCTCGTTACCAAATGT (SEQ ID NO: 121)ApUbx antisense acgagtgctgaagtccctagccaacacgtcTCGTGCTCGTTACCAAATGT (SEQID fwd NO: 122) ApUbx antisenseagaaactagagcttgtcgatcgttaattaaTTTTACCGTCACAGGCATCA (SEQ ID rev NO: 123)ApbAs sense fwd ctacaaatctatctctcctaggGGTGTCACCATCGAGACGTT (SEQ ID NO:124) ApbAs sense revaaaaaccacatacctcgagtggggacgtgtCATGACTCTGGCAGTTGAAGTT (SEQ ID NO: 125)ApbAs antisense aaaaaccacatacctcgagtgggacacgtcCATGACTCTGGCAGTTGAAGTT fwd(SEQ ID NO: 126) ApbAs antisenseagaaactagagcttgtcgatcgttaattaaGGTGTCACCATCGAGACGTT (SEQ ID rev NO: 127)Apcactus sense ctacaaatctatctctcctaggGTCGTCGTCGTCGTCGTAGT (SEQ ID NO:128) fwd Apcactus senseaccaaaattgccttggacagtgggacgtgtGCACGCACGGAAAACATTTA (SEQ ID rev NO: 129)Apcactus accaaaattgccttggacagtggacacgtcGCACGCACGGAAAACATTTA (SEQ IDantisense fwd NO: 130) ApcactusagaaactagagcttgtcgatcgttaattaaGTCGTCGTCGTCGTCGTAGT (SEQ ID antisense revNO: 131)

TABLE 14 List of primers to generate amplicons for the Gibson assemblyto generate hairpin dsRNA sequences against Myzus persicae genes. Eachtarget gene's region will be amplified as either sense or antisense withflanking regions that are homologous to the plasmid backbone for Gibsonassembly. Primer name Primer sequence MpGlnT1 sense fwdctacaaatctatctctcctaggTTGGAAGGGATTGGTGTTGTAATGCC (SEQ ID NO: 132)MpGlnT1 sense revttacaaactgggaagaacctggagacgtgTTCCAGGTTCTTCCCAGTTTGTAACTAGATCG (SEQ IDNO: 133) MpGlnT1 antisensettacaaactgggaagaacctggaacacgtcTCCAGGTTCTTCCCAGTTTGTAACTAGATCG fwd (SEQID NO: 134) MpGlnT1 antisenseagaaactagagcttgtcgatcgttaattaaTTGGAAGGGATTGGTGTTGTAATGCC (SEQ ID rev NO:135) MpUbx sense fwd ctacaaatctatctctcctaggTCGTGTGGAGCAAGTACAGCG (SEQ IDNO: 136) MpUbx sense revacgagtgctgaagtccctagccagacgtgtTGGCTAGGGACTTCAGCACTCG (SEQ ID NO: 137)MpUbx antisense acgagtgctgaagtccctagccaacacgtcTGGCTAGGGACTTCAGCACTCG(SEQ ID fwd NO: 138) MpUbx antisenseagaaactagagcttgtcgatcgttaattaaTCGTGTGGAGCAAGTACAGCGG (SEQ ID NO: rev139) MpbAs sense fwd ctacaaatctatctctcctaggGAGGAACTCCAACTGCCAGAGTCATG(SEQ ID NO: 140) MpbAs sense revaaaaaccacatacctcgagtggggacgtgtCCCACTCGAGGTATGTGGTTTTTCCTATG (SEQ ID NO:141) MpbAs antisenseaaaaaccacatacctcgagtgggacacgtcCCCACTCGAGGTATGTGGTTTTTCC (SEQ fwd ID NO:142) MpbAs antisenseagaaactagagcttgtcgatcgttaattaaGAGGAACTCCAACTGCCAGAGTCATG (SEQ rev ID NO:143) Mpcactus sense fwd ctacaaatctatctctcctaggTACACCCATTGTGTGCACCTGAGTAC(SEQ ID NO: 144) Mpcactus sense revaccaaaattgccttggacagtgggacgtgtCCACTGTCCAAGGCAATTTTGGTTG (SEQ ID NO: 145)Mpcactus antisenseaccaaaattgccttggacagtggacacgtcCCACTGTCCAAGGCAATTTTGGTTG (SEQ ID fwd NO:146) Mpcactus antisenseagaaactagagcttgtcgatcgttaattaaTACACCCATTGTGTGCACCTGAGTAC (SEQ ID rev NO:147)

Once the sense and the antisense amplicons are generated, the plasmidwill be double digested to generate overhangs at the end of the pCaMV35S and the start of the 35S terminator. The digested plasmid, sense,and the antisense amplicons will be assembled together in a Gibsonassembly using Gibson assembly kits (such as SGI Gibson Assembly kit).E. coli (DH5α, electrocompetent cells) will then be transformed with theplasmid and grown on LB plates containing 50 mg/ml kanamycin. Resistantcolonies will be used to harvest the plasmid containing the dsRNAexpression cassette. The plasmids will be named pGlnT1dsRNA, pUbxdsRNA,pbASdsRNA, and pCactdsRNA for each of the four different inserts. Theseplasmids will be used to transform A. tumefaciens via electroporation.Upon selection on LB medium containing Kanamycin (50 mg/ml) andGentamycin (50 mg/ml), resistant colonies will be isolated andmaintained on selection plates.

Transformed A. tumefaciens Infiltrated into N. tabacum:

Transformed A. tumefaciens were grown overnight in LB medium with theselection antibiotics until the OD 600 was 0.6. The cells were pelleteddown, resuspended in infiltration medium (10 mM MES, 150 μMacetosyringone, and 10 mM MgCl2, pH 5.5), and adjusted to an OD600 of0.6. The cells were incubated at room temperature for 2-4 hours. Thecell suspension was infiltrated into healthy N. tabacum leaves. Theinfiltration process was achieved by placing the blunt open end of a 1ml syringe on the underside of the leaf and forcing the cell suspensioninto the leaf. The infiltrated areas were easily distinguished from theuntreated areas and were clearly demarcated using a marker. The plantswere then covered to created high moisture environment for 24 h.

The leaves expressed green fluorescent protein and were visualized underan epi fluorescence microscope after 1-2 days, see FIG. 19. The geneticmaterial that was transferred to the plant by the A. tumefacienscontained a green fluorescence (GFP) expression cassette that was drivenby an ubiquitin promoter that was active in N. tabacum. The expressionof GFP will be used a proxy for the expression of dsRNA.

Generating a Stable Clone of N. tabacum Expressing dsRNA

The infiltrated leaves that show expression of GFP will be isolated, andthe regions of the leaf near the midrib that express GFP very stronglywill be cutout. The leaf disks will then be thoroughly sterilized usinga sterilization solution (2% hypochlorite, 0.01% tween 20) for 10 min byagitation. The sterile leaf disks will be placed on petri dishescontaining shooting medium (2.15 g/I Murashige and Skoog salts (withoutIAA, kinetin or sucrose), 0.8% (w/v) agar, 3.0% (w/v) sucrose, 0.1 mg/lindole butyric acid, 0.8 mg/l 6-benzylaminopurine, 0.1 mg/lCarbenicillin, 0.2 mg/l Ticarcillin/Clavulanic acid and suitableselection for the binary vector carrying your FP fusion construct) at25° C., 16 h:8 h light:dark cycle till shoots appear. The new shootswill then be transplanted into plates containing rooting medium (2.15g/I Murashige and Skoog salts, 0.8% (w/v) agar, 3.0% (w/v) sucrose, 0.5mg/l indole butyric acid, 0.1 mg/l carbenicillin, 0.2 mg/lTicarcillin/Clavulanic acid) at 25° C., 16 h:8 h light:dark cycle tillroots appear. These new plants will be transferred to phytatrays todevelop larger roots so that they can be transferred to soil. Theexpression of the GFP will be tested in all new clones to ensure stableexpression.

Treating Aphids with dsRNA by Rearing them on N. tabacum ExpressingdsRNA

Aphids will be grown on 10-week-old N. tabacum plants in a climatecontrolled incubator (16 h:8 h light:dark cycle, 60% humidity, 25° C.).To limit maternal effects or health differences between plants, 5-10adults from different plants will be distributed among 10 two-week-oldplants and allowed to multiply to high density for 5-7 days. Forexperiments, first and second instar aphids will be collected fromhealthy plants and divided into two different treatment groups: 1) thoseallowed to feed on leaves that express the dsRNA, and 2) those allowedto feed on control leaves that do no express the dsRNA.

For each feeding experiment, leaves will be taken from the plant andplaced in a 1.5 ml Eppendorf sealed with parafilm. The leaf stem will beplaced into a deep petri dish (Fisher Scientific, Cat# FB0875711) andaphids will be applied to the leaves of the plant and allowed to feed.Old leaves will be replaced with new, freshly injected leaves every 2-3days throughout the experiment. For each treatment, 60 aphids will beplaced onto each leaf. Aphids will be monitored daily for survival anddead aphids will be removed when they were discovered. In addition, thedevelopmental stage (1st, 2nd, 3rd, 4th, and 5th instar) will bedetermined daily throughout the experiment.

After 5 and 6 days of treatment, DNA will be extracted from dead aphidsfrom each treatment group. Briefly, the aphid body surface will besterilized by dipping the aphid into a 6% bleach solution forapproximately 5 seconds. Aphids will then be rinsed in sterile water andDNA will be extracted from each individual aphid using a DNA extractionkit (Qiagen, DNeasy kit) according to manufacturer's instructions. DNAconcentration will be measured using a nanodrop nucleic acidquantification, and Buchnera and aphid DNA copy numbers will be measuredby qPCR. The primers that will be used for Buchnera are Buch_groES_18F(CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 101) and Buch_groES_98R(CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 102) (Chong and Moran, 2016 PNAS).The primers that will be used for aphid are ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 103) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 104) (Chong and Moran, 2016 PNAS).qPCR will be performed using a qPCR amplification ramp of 1.6 degreesC./s and the following conditions: 1) 95° C. for 10 minutes, 2) 95° C.for 15 seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5)95° C. for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15degrees C./s, 8) 95° C. for 1 second. qPCR data will be analyzed usinganalytic (Thermo Fisher Scientific, QuantStudio Design and Analysis)software.

At 7 post-treatment, RNA will be extracted from live aphids and RT-pPCRwill be performed to quantify expression of BCR-4. Briefly, the aphidbody surface will be sterilized by dipping the aphid into a 6% bleachsolution for approximately 5 seconds. Aphids will then be rinsed insterile water and total RNA will be extracted from each individual aphidusing the RNA extraction kit (Qiagen miRNeasy kit) according tomanufacturer's instructions. RNA concentrations will be measured using ananodrop nucleic acid quantification. BCR-4 relative expression will bemeasured by RT-qPCR. The primers used will be ApBCR-4F(CTCTGTCAACCACCATGAGATTA; SEQ ID NO: 107) and ApBCR-4R(TGCAGACTACAGCACAATACTT; SEQ ID NO: 108). The internal reference geneprimers were for Actin (housekeeping gene). The forward sequence isGATCAGCAGCCACACACAAG; SEQ ID NO: 109 and the reverse sequence isTTTGAACCGGTTTACGACGA; SEQ ID NO: 110. RT-qPCR will be performed using aqPCR amplification ramp of 1.6 degrees C./s and the followingconditions: 1) 48° C. for 30 min, 2) 95° C. for 10 minutes, 3) 95° C.for 15 seconds, 4) 60° C. for 1 minute, 5) repeat steps 3-4 40×, 6) 95°C. for 15 seconds, 7) 60° C. for 1 minute, 8) ramp change to 0.15degrees C./s, 9) 95° C. for 1 second. RT-qPCR data was analyzed usinganalytic (Thermo Fisher Scientific, QuantStudio Design and Analysis)software.

As shown in previous Examples, the aphids fed on the leaves expressingthe dsRNA against the aphid target genes are expected to have lowersurvival, develop slower, contain fewer Buchnera, and have reducedtarget gene expression compared to the aphids reared on the controlleaves.

Together this data described herein demonstrate the ability to kill anddecrease the development and longevity (e.g., fitness) of aphids bytreating them with plants expressing dsRNA targeting the essentialgene(s) (e.g. glutamine transporter ApGLNT1) of bacteriocytes in theaphids.

Other Embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A method for decreasing the fitness of an agricultural insect pest,the method comprising: (a) providing a composition comprising (i)validamycin and (ii) a modulating agent, wherein the modulating agentdisrupts an interaction between the agricultural insect pest and aresident microorganism thereof; and (b) delivering said composition tothe agricultural insect pest, whereby the fitness of the agriculturalinsect pest treated with the composition is decreased relative to anuntreated agricultural insect pest.
 2. The method of claim 1, whereinthe modulating agent is a polypeptide, a small molecule, or a nucleicacid.
 3. The method of claim 1, wherein the modulating agent targets apathway in the agricultural insect pest that mediates an interactionbetween the agricultural insect pest and the resident microorganismthereof.
 4. The method of claim 1, wherein the modulating agent targetsa pathway in the resident microorganism that mediates an interactionbetween the agricultural insect pest and the resident microorganismthereof.
 5. The method of claim 1, wherein the level of the residentmicroorganism in the agricultural insect pest is decreased relative toan untreated agricultural insect pest.
 6. The method of claim 1, whereinthe metabolism of the resident microorganism in the agricultural insectpest is decreased relative to an untreated agricultural insect pest. 7.The method of claim 1, wherein the population of the residentmicroorganism in the agricultural insect pest is decreased by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to an untreatedagricultural insect pest.
 8. The method of claim 1, wherein thediversity of resident microorganisms in the agricultural insect pest isreduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%relative to an untreated agricultural insect pest.
 9. The method ofclaim 1, wherein the decrease in fitness of the agricultural insect pestis measured as a decrease in a physiological parameter of the insect.10. The method of claim 1, wherein the decrease in fitness of theagricultural insect pest is measured as death of the insect.
 11. Themethod of claim 1, wherein the delivery comprises delivering thecomposition to at least one habitat where the agricultural insect pestgrows, lives, reproduces, feeds, or infests.
 12. The method of claim 1,wherein the composition is formulated with an agriculturally acceptablecarrier as a liquid, a solid, an aerosol, a paste, a gel, or a gascomposition.
 13. The method of claim 1, wherein the composition isdelivered as a spray to an agricultural crop.
 14. The method of any oneof claims 1-13, wherein the resident microorganism is a symbiont of theagricultural insect pest.
 15. The method of claim 14, wherein thesymbiont is an endosymbiont.
 16. The method of claim 14 or 15, whereinthe symbiont is an obligate symbiont or a facultative symbiont.
 17. Themethod of any one of claims 1-13, wherein the resident microorganism isa commensal microorganism of the agricultural insect pest.
 18. Themethod of claim 1, wherein the agricultural insect pest is an aphid, astinkbug, or a whitefly.
 19. The method of claim 18, wherein theagricultural insect pest is an aphid.
 20. The method of claim 19,wherein the aphid is Acyrthosiphon pisum.
 21. The method of claim 19 or20, wherein the resident microorganism is Buchnera aphidicola.
 22. Themethod of claim 18, wherein the agricultural insect pest is a stinkbug.23. The method of claim 22, wherein the stinkbug is a Nezara species oran Oebalus species.
 24. The method of claim 22 or 23, wherein theresident microorganism is a Pantoea species.
 25. The method of claim 18,wherein the agricultural insect pest is a whitefly.
 26. The method ofclaim 25, wherein the whitefly is Bemisia tabaci.
 27. The method ofclaim 25 or 26, wherein the resident microorganism is Portieraaleyrodidarum.